Gene therapy vectors for treating heart disease

ABSTRACT

The present disclosure provides methods and compositions useful for the treatment or prevention of heart disease. In particular, the present disclosure provides a vector comprising a modified troponin T promoter operatively linked to a therapeutic gene product for the treatment or prevention of heart disease, e.g., cardiomyopathy. The gene product may be MYBPC3. The disclosure also provides recombinant adeno-associated virus (rAAV) virions, rAAV viral genomes, and expression cassettes and pharmaceutical compositions thereof. The disclosure further provides methods for treating a disease or disorder, such as heart disease.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.17/210,882, filed Mar. 24, 2021, which is a continuation ofInternational PCT Application No. PCT/US2021/017699, filed Feb. 11,2021, which claims the benefit of priority to U.S. Provisional PatentApplication Ser. No. 63/047,633 filed on Jul. 2, 2020 and U.S.Provisional Patent Application Ser. No. 62/976,160 filed on Feb. 13,2020, the contents of which are hereby incorporated by reference intheir entireties.

TECHNICAL FIELD

The present disclosure relates to compositions and methods for thetreatment or prevention of heart disease (e.g., cardiomyopathy) in asubject. In particular, the present disclosure relates to a vectorcomprising a cardiac-specific promoter operability linked to atherapeutic gene product for the treatment of heart disease (e.g.,cardiomyopathy).

REFERENCE TO SEQUENCE LISTING

This application is being filed electronically via EFS-Web and includesan electronically submitted sequence listing in .txt format. The .txtfile contains a sequence listing entitled“TENA_015_03US_SeqList_ST25.txt” created on Jul. 22, 2021 and having asize of 824 kilobytes. The sequence listing contained in this .txt fileis part of the specification and is incorporated herein by reference inits entirety.

BACKGROUND

Gene therapy approaches for the treatment of heart disease often employvectors configured to effectively transduce cardiac cells and to expressa transgene in a cardiac-tissue specific manner. Adeno-associated virus(AAV) vectors, cardiac-specific promoters, or both in combination, maybe used to deliver a polynucleotide encoding a gene product (e.g. atherapeutic protein) to heart tissue and thereby express the geneproduct in that tissue to treat the heart disease. Cardiac-specificpromoters include desmin (Des), alpha-myosin heavy chain (α-MHC), myosinlight chain 2 (MLC-2) and cardiac troponin C (TNNC1 or cTnC) promoters,as well as the 600 base pair cardiac troponin T (TNNT2) promoter. Thedelivery of polynucleotides encoding large proteins remains challenging,however, due in part to the packaging limit of viral vectors.

Given these challenges, there remains a need in the art for improvedgene therapy vectors for heart disease.

SUMMARY

The present disclosure relates generally to compositions and methods forthe treatment or prevention of heart disease (e.g. cardiomyopathy). In afirst aspect, the present disclosure provides vectors comprising apromoter, optionally a cardiac-specific promoter, operably linked to apolynucleotide encoding a therapeutic gene product for the treatment ofprevention of heart disease, e.g., cardiomyopathy. The vector may be anadeno-associated viral (AAV) vector.

In some aspects, the present disclosure provides a cardiac troponin Tpromoter, comprising a polynucleotide having between 300 bp and 500 bp.In some embodiments, the polynucleotide comprises a sequence that sharesat least 80%, at least 90%, or at least 100% identity to any one of SEQID NOs: 1-85. In some embodiments, the polynucleotide comprises asequence that shares at least 80%, at least 90%, or at least 100%identity to SEQ ID NO: 1. In some embodiments, the polynucleotidecomprises a sequence that shares at least 80%, at least 90%, or at least100% identity to SEQ ID NO: 3. In some embodiments, the polynucleotideshares at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or and 100% sequenceidentity with a genomic polynucleotide sequence upstream of andincluding the transcription start site of a troponin T gene. In someembodiments, the polynucleotide shares at least 80%, 90%, 95%, 96%, 97%,98%, 99%, or and 100% sequence identity with a genomic polynucleotidesequence −450 bp to +1 bp relative to the transcription start site of atroponin T gene. In some embodiments, the polynucleotide shares at least80%, 90%, 95%, 96%, 97%, 98%, 99%, or and 100% sequence identity with agenomic polynucleotide sequence −350 bp to +1 bp relative to thetranscription start site of a troponin T gene. In some embodiments, thepolynucleotide shares at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or and100% sequence identity with a genomic polynucleotide sequence −250 bp to+1 bp relative to the transcription start site of a troponin T gene. Insome embodiments, the polynucleotide shares at least 80%, 90%, 95%, 96%,97%, 98%, 99%, or and 100% sequence identity with a genomicpolynucleotide sequence −450 bp to +50 bp relative to the transcriptionstart site of a troponin T gene. In some embodiments, the polynucleotideshares at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or and 100% sequenceidentity with a genomic polynucleotide sequence −350 bp to +50 bprelative to the transcription start site of a troponin T gene. In someembodiments, the polynucleotide shares at least 80%, 90%, 95%, 96%, 97%,98%, 99%, or and 100% sequence identity with a genomic polynucleotidesequence −250 bp to +50 bp relative to the transcription start site of atroponin T gene. In some embodiments, the troponin T gene is a humantroponin T gene.

In some embodiments, the promoter is a muscle-specific promoter. In someembodiments, the promoter is a cardiac cell-specific promoter. In someembodiments, the promoter is a cardiomyocyte-specific promoter. In someembodiments, the promoter has the same cell-type specificity as a nativetroponin T promoter of about 600 bp. In some embodiments, the promoterdescribed herein has the same cell-type specificity as a referencepromoter comprising SEQ ID NO: 1. In some embodiments, the promoterexpresses a gene product operatively linked thereto at least about 10%,at least about 20%, at least about 30% more than a native troponin Tpromoter. In some embodiments, the promoter described herein expresses agene product operatively linked thereto at least about 10%, at leastabout 20%, at least about 30% more than a reference promoter comprisingSEQ ID NO: 1.

In some aspects, the present disclosure provides a vector comprising anyone of the promoters described herein operatively linked to apolynucleotide encoding a gene product. In some embodiments the vectoris a viral vector. In some embodiments, the viral vector is anadeno-associated virus vector (AAV). In some embodiments, the viralvector has a packaging limit of at most about 5.5 kb.

In some embodiments, the gene product is selected from MYBPC3, KCNH2,TRPM4, DSG2, and ATP2A2 protein. In some embodiments, the gene productis selected from CACNA1C, DMD, DMPK, EPG5, EVC, EVC2, FBN1, NF1, SCN5A,SOS1, NPR1, ERBB4, VIP, and MYH7 proteins. In some embodiments, the geneproduct is a Cas9, optionally selected from SpCas9, St1Cas9, and SaCas9.

In some embodiments, the vector described herein comprises apolynucleotide encoding a second gene product. In some embodiments, thesecond gene product is a functional RNA, optionally a microRNA or aguide RNA.

In some aspects, the present disclosure provides an isolated cellcomprising any one of the promoters described herein. In someembodiments, the isolated cell is an induced pluripotent stem cell or anisolated cardiomyocyte.

In some aspects, the present disclosure provides a pharmaceuticalcomposition comprising any one of the vectors described herein.

In some aspects, the present disclosure provides a cell therapycomposition comprising any one of the isolated cells described herein.

In some aspects, the present disclosure provides a recombinantadeno-associated virus (AAV) vector genome, comprising a MYBPC3polynucleotide encoding a MYBPC3 protein, or a functional variantthereof, and a promoter, wherein the promoter is a polynucleotide havingbetween 300 bp and 500 bp. In some embodiments, the polynucleotidecomprises a sequence that shares at least 80%, at least 900, or at least100% identity to any one of SEQ ID NOs: 1-85. In some embodiments, thepolynucleotide comprises a sequence that shares at least 80%, at least90%, or at least 100% identity to any one of SEQ ID NO: 1. In someembodiments, the polynucleotide comprises a sequence that shares atleast 80%, at least 90%, or at least 100% identity to any one of SEQ IDNO: 3. In some embodiments, the polynucleotide shares at least 80%, 90%,95%, 96%, 97%, 98%, 99%, or and 100% sequence identity with a genomicpolynucleotide sequence upstream of and including the transcriptionstart site of a troponin T gene. In some embodiments, the polynucleotideshares at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or and 100% sequenceidentity with a genomic polynucleotide sequence −450 bp to +1 bprelative to the transcription start site of a troponin T gene. In someembodiments, the polynucleotide shares at least 80%, 90%, 95%, 96%, 97%,98%, 99%, or and 100% sequence identity with a genomic polynucleotidesequence −350 bp to +1 bp relative to the transcription start site of atroponin T gene. In some embodiments, the polynucleotide shares at least80%, 90%, 95%, 96%, 97%, 98%, 99%, or and 100% sequence identity with agenomic polynucleotide sequence −250 bp to +1 bp relative to thetranscription start site of a troponin T gene. In some embodiments, thepolynucleotide shares at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or and100% sequence identity with a genomic polynucleotide sequence −450 bp to+50 bp relative to the transcription start site of a troponin T gene. Insome embodiments, the polynucleotide shares at least 80%, 90%, 95%, 96%,97%, 98%, 99%, or and 100% sequence identity with a genomicpolynucleotide sequence −350 bp to +50 bp relative to the transcriptionstart site of a troponin T gene. In some embodiments, the polynucleotideshares at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or and 100% sequenceidentity with a genomic polynucleotide sequence −250 bp to +50 bprelative to the transcription start site of a troponin T gene. In someembodiments, the troponin T gene is a human troponin T gene.

In some embodiments, the promoter is a muscle-specific promoter. In someembodiments, the promoter is a cardiac cell-specific promoter. In someembodiments, the promoter is a cardiomyocyte-specific promoter. In someembodiments, the promoter has the same cell-type specificity as a nativetroponin T promoter of about 600 bp. In some embodiments, the promoterhas the same cell-type specificity as a reference promoter comprisingSEQ ID NO: 1. In some embodiments, the promoter expresses a gene productoperatively linked thereto at least about 10%, at least about 20%, atleast about 30% more than a native troponin T promoter. In someembodiments, the promoter expresses a gene product operatively linkedthereto at least about 10%, at least about 20%, at least about 30% morethan a reference promoter comprising SEQ ID NO. 1.

In some embodiments, the recombinant adeno-associated virus (AAV) vectorgenome described herein comprises a MYBPC3 polynucleotide encoding aMYBPC3 protein. In some embodiments, the MYBPC3 polynucleotide comprisesat least about 3.5 kB. In some embodiments, the MYBPC3 polynucleotidecomprises about 3.8 kB. In some embodiments, the MYBPC3 is a full-lengthMYBPC3. In some embodiments, the MYBPC3 is a truncated MYBPC3.

In some embodiments, the rAAV vector genome described herein expressesMYBPC3. In some embodiments, the rAAV vector genome expresses MYBPC3 atabout the same level as a reference AAV vector comprising a nativetroponin T promoter of about 600 bp. In some embodiments, the rAAVvector genome expresses MYBPC3 at a level at least about 10% greaterthan a reference AAV vector comprising a native troponin T promoter ofabout 600 bp. In some embodiments, the rAAV vector genome expressesMYBPC3 at a level at least about 20% greater than a reference AAV vectorcomprising a native troponin T promoter of about 600 bp.

In some aspects, the present disclosure provides a recombinantadeno-associated virus (AAV) vector genome, comprising an expressioncassette comprising, in 5′ to 3′ order, a 5′ segment comprising apromoter; a polynucleotide encoding a gene product; and a 3′ segmentcomprising a polyA signal, the expression cassette optionally flanked byone or both of a 5′ inverted terminal repeat (ITR) and a 3′ ITR, whereinthe polynucleotide encoding the gene product comprises between 3 kb and11 kb, between 3 kb and 5 kb, between 3.5 kb and 4.5 kb, or between 3.7kb and 4 kb; and wherein: a) the 5′ segment and the 3′ segment togethercomprise at most 0.8 kbp or at most 0.9 kbp; b) the 5′ ITR, 5′ segment,the 3′ segment, and 3′ ITR together comprise or at most 1.2 kbp, at most1.3 kbp; and/or c) the vector genome comprises at most 4.7 kbp, at most4.8 kbp, at most 4.9 kbp, or at most 5.0 kbp. In some embodiments, the5′ segment comprises at most 500 bp or at most 480 bp. In someembodiments, the 3′ segment comprises at most 200 bp or at most 150 bp.

In some embodiments, the rAAV vector genome comprises a polynucleotideencoding a gene product comprising 3.7 kbp to 3.9 kbp, optionally 3.8kbp. In some embodiments, the gene product is MYBPC3, or a functionalvariant thereof. In some embodiments, the gene product is MYBPC3. Insome embodiments, the polynucleotide encoding MYBPC3 shares at least 90%identity to SEQ ID NO: 86. In some embodiments, the polynucleotideencoding MYBPC3 share at least 95% identify to SEQ ID NO: 86. In someembodiments the polynucleotide encoding MYBPC3 is SEQ ID NO: 86. In someembodiments, MYBPC3 shares at least 90% identity to the polypeptidesequence of SEQ ID NO: 103. In some embodiments, MYBPC3 shares at least95% identity to the polypeptide sequence of SEQ ID NO: 103. In someembodiments, MYBPC3 shares 100% identity to the polypeptide sequence ofSEQ ID NO: 103.

In some embodiments, the rAAV vector genome comprises a promoter,wherein the promoter is a polynucleotide having between 300 bp and 500bp. In some embodiments the promoter comprises a sequence that shares atleast 80% identity to SEQ ID NO:1-85. In some embodiments the promotercomprises a sequence that shares at least 90% identity to SEQ IDNO:1-85. In some embodiments the promoter comprises a sequence thatshares at least 100% identity to SEQ ID NO:1-85.

In some embodiments, the rAAV vector genome comprises a polyA signal. Insome embodiments, the polyA signal comprises, consists essentially of,or consists of a sequence that shares at least 90% identity to SEQ IDNO: 92. In some embodiments, the polyA signal comprises, consistsessentially of, or consists of a sequence that shares at least 95%identity to SEQ ID NO: 92. In some embodiments, the polyA signal is SEQID NO: 92.

In some embodiments, the rAAV vector genome comprises a 5′ segment. Insome embodiments, the 5′ segment shares at least 80% identity to SEQ IDNO: 93. In some embodiments, the 5′ segment shares at least 90% identityto SEQ ID NO: 93. In some embodiments, the 5′ segment shares at least95% identity to SEQ ID NO: 93. In some embodiments, the 5′ segment isSEQ ID NO: 93.

In some embodiments, the rAAV vector genome comprises a 3′ segment. Insome embodiments, the 3′ segment shares at least 80% identity to SEQ IDNO: 94. In some embodiments, the 3′ segment shares at least 90% identityto SEQ ID NO: 94. In some embodiments, the 3′ segment shares at least95% identity to SEQ ID NO: 94. In some embodiments, the 3′ segment isSEQ ID NO: 94.

In some embodiments, the rAAV vector genome comprises an expressioncassette. In some embodiments, the expression cassette shares at least80% identity to SEQ ID NO: 95. In some embodiments, the expressioncassette shares at least 90% identity to SEQ ID NO: 95. In someembodiments, the expression cassette shares at least 95% identity to SEQID NO: 95. In some embodiments, the expression cassette is SEQ ID NO:95.

In some embodiments, the rAAV genome comprises an expression cassettethat is flanked by one or both of a 5′ inverted terminal repeat (ITR)and a 3′ ITR. In some embodiments, the 5′ ITR comprises a sequence thatshares 95% identity to SEQ ID NO: 96. In some embodiments, the 3′ ITRcomprises a sequence that shares at least 95% identity to SEQ ID NO: 97.

In some aspects, the present disclosure provides a recombinant AAV(rAAV) virion. In some embodiments the rAAV virion comprises any one ofthe rAAV vector genomes described herein and an AAV capsid protein.

In some aspects, the present disclosure provides a method of expressinga MYBPC3 protein in a cell, comprising transducing the cell with an rAAVvirion described herein or any one of the rAAV vector genomes describedherein. In some embodiments, the cell is a MYBPC3^(−/−) cell. In someembodiments, the cell comprises an inactivating mutation in one or bothcopies of the endogenous MYBPC3 gene.

In some aspects, the present disclosure provides a method of treatingand/or preventing a cardiomyopathy in a subject in need thereof,comprising administering the rAAV virion described herein or any one ofthe rAAV vector genomes described herein to the subject, whereinoptionally the subject suffers from or is at risk for cardiomyopathy.

In some aspects, the present disclosure provides a method of expressinga MYBPC3 protein in the heart of a subject in need thereof, comprisingadministering the rAAV virion described herein or any one of the rAAVvector genomes described herein to the subject, optionally a subjectsuffering from or at risk for cardiomyopathy, wherein optionally thesubject suffers from or is at risk for cardiomyopathy.

In some embodiments, administration of the AAV vector causes specificexpression of MYBPC3 in the heart of the subject. In some embodiments,administration of the AAV vector causes low or undetectable expressionof MYBPC3 in the skeletal tissue, brain, and/or liver of the subject,wherein optionally the subject suffers from or is at risk forcardiomyopathy.

In some aspects, the present disclosure provides a method of treating adisease caused by a MYBPC3 mutation in a subject in need thereof,comprising administering the rAAV virion described or any one of therAAV vector genomes described herein to the subject, wherein optionallythe subject suffers from or is at risk for cardiomyopathy.

In some aspects, the present disclosure provides a method of increasingMYBPC3 activity and/or increasing cardiac function in the heart of asubject in need thereof, comprising administering the rAAV viriondescribed herein or any one of the rAAV vector genomes described hereinto the subject, wherein optionally the subject suffers from or is atrisk for cardiomyopathy.

In some embodiments, the methods described herein treats thecardiomyopathy. In some embodiments, the methods described hereinprevents the cardiomyopathy. In some embodiments, the cardiomyopathy ishypertrophic cardiomyopathy.

In some embodiments, the methods described herein comprise intravenousadministration of the rAAV virion described herein or any one of therAAV vector genomes described herein to the subject. In someembodiments, the methods described herein comprise intracardiacadministration of the rAAV virion described herein or any one of therAAV vector genomes described herein to the subject. In someembodiments, the methods described herein comprise direct injection ofthe rAAV virion described herein or any one of the rAAV vector genomesdescribed herein to the subject. In some embodiments, the methodsdescribed herein comprise administering a dose of about 10¹¹ to about10¹⁴ rAAV virions per kg or viral genomes per kg.

In some embodiments, the subject is a mammal. In some embodiments, thesubject is a human. In some embodiments, the subject is an adult.

In some embodiments, the pharmaceutical compositions described hereinare for use as a medicament in therapeutic or prophylactic treatment ofheart disease, e.g. cardiomyopathies.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows maps of insert sequences for AAV vector genomes adaptedfor large cargoes, showing deletion or truncation of two cis-regulatoryelements.

FIG. 1B shows flow cytometry analysis, two days post-infection, of humancardiac fibroblasts (n=2), MOI 160,000 with AAV-packaged constructs.

FIG. 1C shows a map of an insert sequence for an AAV vector genomeadapted for large cargoes, showing deletion or truncation of twocis-regulatory elements, deletion of the intron, and partial deletion ofthe sequence 3′ to the 5′ ITR.

FIG. 2A shows a schematic of the original and altered versions of aviral genome comprising the cardiac-specific troponin (TNNT2) promoterand myosin binding protein C (MYBPC3) transgene.

FIG. 2B shows detection of MYBPC3 protein by immunofluorescence inMYBPC3^(−/−) iPSC-derived cardiomyocytes transduced with AAV6-packagedconstructs encoding MYBPC3 driven by the cardiac-specific TNNT2promoter.

FIG. 3A shows detection of MYBPC3 protein by Western blot inMYBPC3^(−/−) PSC-derived cardiomyocytes transduced with AAV6-packagedconstructs encoding human MYBPC3 driven by various sizes (400-600 bp) ofthe cardiac-specific TNNT2 promoter. GAPDH was used as a loadingcontrol.

FIG. 3B shows detection of MYBPC3 protein by Western blot inMYBPC3^(−/−) iPSC-derived cardiomyocytes transduced with AAV6-packagedconstructs encoding human MYBPC3 driven by various sizes (400 or 600 bp)of the human cardiac TNNT2 promoter. No Kozak sequence was used as anegative control and GAPDH was used as a loading control.

FIG. 3C shows detection of MYBPC3 protein by Western blot inMYBPC3^(−/−) iPSC-derived cardiomyocytes transfected with AAV6 plasmidsencoding human MYBPC3 driven by various sizes (400 or 600 bp) of thehuman cardiac TNNT2 promoter. GAPDH was used as a loading control.

FIG. 4A shows a map of the introns of the Mybpc3 gene and a dot blot ofMYBPC3 protein expression in the founder mouse of the KO line.

FIG. 4B shows a bar graph of body weight of wild-type (WT) or KO mice(Mybpc3^(−/−)) littermates at two weeks of age. WT (n=11), Mybpc3^(−/−)(n=7) and Mybpc3^(−/−) (n=13).

FIG. 4C shows a bar graph of ejection fraction (%) measured byechocardiography in wild-type (WT), heterozygous KO mice (Mybpc3^(−/−)),or homozygous KO mice (Mybpc3^(−/−)) at two weeks of age.

FIG. 4D shows a bar graph of fractional shortening measured byechocardiography in wild-type (WT), heterozygous KO mice (Mybpc3^(−/−)),or homozygous KO mice (Mybpc3^(−/−)) at two weeks of age.

FIG. 4E shows a bar graph of left ventricular (LV) mass normalized bybody weight (BW) in wild-type (WT), heterozygous KO mice (Mybpc3^(−/−)),or homozygous KO mice (Mybpc3^(−/−)) at two weeks of age.

FIG. 4F shows a bar graph of Left ventricular internal diameter duringsystole (LVIDs) normalized by body weight in wild-type (WT),heterozygous KO mice (Mybpc3^(−/−)), or homozygous KO mice(Mybpc3^(−/−)) at two weeks of age.

FIG. 4G shows a bar graph of Left ventricular internal diameter duringdiastole (LVIDd) normalized by body weight in wild-type (WT),heterozygous KO mice (Mybpc3^(−/−)), or homozygous KO mice(Mybpc3^(−/−)) at two weeks of age.

FIG. 5 shows detection of MYBPC3 mRNA by qRT-PCR in heart, skeletal,brain and liver tissue harvested from mice retro-orbitally injected withE12 GC AAV9-packaged constructs encoding human MYBPC3 driven by varioussizes (400 or 600 bp) of the human cardiac TNNT2 promoter.

FIG. 6A shows a bar graph showing absolute quantification of vectorgenomes per microgram of genomic DNA in the heart and liver of adultmice 4 weeks after intravenous administration with an AAV9 vectorcontaining the 400 bp modified TNNT2 promoter cassette.

FIG. 6B shows a bar graph showing fold increase over vehicle oftransgene RNA in the heart and liver of adult mice 4 weeks afterintravenous administration with an AAV9 vector containing the 400 bpmodified TNNT2 promoter cassette.

FIG. 7 shows a western blot of MYBPC3 protein expression in homozygousMybpc3^(−/−) mice injected retro-orbitally at two weeks of age with 1E14vg·kg⁻¹ test vector encoding Mybpc3 or vehicle, HBSS.

FIG. 8 shows a bar graph showing MYBPC3 expression in homozygousMybpc3^(−/−) mice injected retro-orbitally at two weeks of age 3E13vg·kg⁻¹ and 1E14 vg·kg⁻¹ test vector encoding Mybpc3 or vehicle, HBSS.

FIG. 9A shows a bar graph showing left ventricular mass normalized tobody weight (LVM/BM) in homozygous Mybpc3^(−/−) mice 6 weeks after theywere injected retro-orbitally at two weeks of age with 1E13 vg·kg-1,3E13 vg·kg⁻¹, and 1E14 vg·kg⁻¹ of test vector encoding Mybpc3 orvehicle, HBSS.

FIG. 9B shows a bar graph showing FAS expressed as % percentage changein LV internal dimensions between systole and diastole in homozygousMybpc3^(−/−) mice 6 weeks after they were injected retro-orbitally attwo weeks of age with 1E13 vg·kg⁻¹, 3E13 vg·kg⁻¹, and 1E14 vg·kg-1 oftest vector encoding Mybpc3 or vehicle, HBSS.

FIG. 9C shows a bar graph showing ejection fraction in homozygousMybpc3^(−/−) mice 6 weeks after they were injected retro-orbitally attwo weeks of age with 1E13 vg·kg⁻¹, 3E13 vg·kg⁻¹, and 1E14 vg·kg-1 oftest vector encoding Mybpc3 or vehicle, HBSS.

FIG. 9D shows a bar graph showing left ventricular mass normalized tobody weight (LVM/BM) in homozygous Mybpc3^(−/−) mice 31 weeks after theywere injected retro-orbitally at two weeks of age with 1E13 vg·kg-1,3E13 vg·kg⁻¹, and 1E14 vg·kg⁻¹ of test vector encoding Mybpc3 orvehicle, HBSS.

FIG. 9E shows a bar graph showing FAS expressed as % percentage changein LV internal dimensions between systole and diastole in homozygousMybpc3^(−/−) mice 31 weeks after they were injected retro-orbitally attwo weeks of age with 1E13 vg·kg⁻¹, 3E13 vg·kg⁻¹, and 1E14 vg·kg⁻¹ oftest vector encoding Mybpc3 or vehicle, HBSS.

FIG. 9F shows a bar graph showing ejection fraction in homozygousMybpc^(−/−) mice 31 weeks after they were injected retro-orbitally attwo weeks of age with 1E13 vg·kg⁻¹, 3E13 vg·kg⁻¹, and 1E14 vg·kg⁻¹ oftest vector encoding Mybpc3 or vehicle, HBSS.

FIG. 10A is an illustration of an AAV9 vector encoding Mybpc3 in thecontext of the 5.4 kbp or 4.7 kbp expression cassettes.

FIG. 10B is a bar graph showing ejection fraction in homozygousMybpc3^(−/−) mice 18 weeks after they were injected retro-orbitally atthree months of age with 3E13 vg·kg⁻¹ or 1E14 vg·kg⁻¹ of AAV9 vectorencoding Mybpc3 in the context of the 5.4 kbp or 4.7 kbp cassettes, orinjected with vehicle control, HBSS.

FIG. 10C is a plot showing ejection fraction progression in homozygousMybpc3^(−/−) mice after they were injected retro-orbitally at threemonths of age with 3E13 vg·kg⁻¹ or 1E14 vg·kg⁻¹ of AAV9 vector encodingMybpc3 in the context of the 5.4 kbp or 4.7 kbp cassettes, or injectedwith vehicle control, HBSS.

FIG. 10D is a bar graph showing left ventricular mass normalized to bodyweight (LVM/BM) in homozygous Mybpc3^(−/−) mice 18 weeks after they wereinjected retro-orbitally at three months of age with 3E13 vg·kg⁻¹ or1E14 vg·kg⁻¹ of AAV9 vector encoding Mybpc3 in the context of the 5.4kbp or 4.7 kbp cassettes, or injected with vehicle control, HBSS.

FIG. 11A is a bar graph showing GFP expression in cardiac tissuefollowing systemic delivery of an AAV9 capsid variant, CR9-10, with aGFP-encoding cassette or AAV9 with a GFP-encoding cassette in adult mice(p<0.05, One-way ANOVA; Dunnett's multiple comparison test).

FIG. 11B is a bar graph showing ejection fraction in Mybpc3^(−/−) miceinjected retro-orbitally at two weeks of age with 1E13 vg·kg⁻¹ and 3E13vg·kg⁻¹ of AAV9 vector encoding Mybpc3, CR9-10 vector encoding Mybpc3,or vehicle control HBSS.

FIG. 11C is a bar graph showing ejection fraction compared to pre-dosebaseline (AEF) in Mybpc3^(−/−) mice injected retro-orbitally at twoweeks of age with 1E13 vg·kg-1 and 3E13 vg·kg-1 of AAV9 vector encodingan expression cassette Mybpc3 gene, CR9-10 vector encoding an expressioncassette Mybpc3 gene, or vehicle control HBSS.

FIG. 12A is a bar graph showing GFP expression in the left ventricle ofnon-human primates one month after intravenous delivery of 1E13 vg·kg-1dose of AAV vector encoding GFP packaged in one of fourteen differentcapsid proteins.

FIG. 12B is a bar graph showing GFP expression in the liver of non-humanprimates one month after intravenous delivery of 1E13 vg·kg-1 dose ofAAV vector encoding GFP packaged in one of fourteen different capsidproteins.

FIG. 12C is a bar graph showing the ratio of GFP expression in the leftventricle:liver of non-human primates one month after intravenousdelivery of 1E13 vg·kg⁻¹ dose of AAV vector encoding GFP packaged in oneof fourteen different capsid proteins.

FIG. 13A is a plot showing ejection fraction progression in homozygousMybpc^(−/−) mice that were injected retro-orbitally at two weeks of agewith AAV9 encoding the mouse Mybpc3 gene (mMybpc3) (at 1E14 vg·kg⁻¹),AAV9 encoding the human MYBPC3 gene (hMYBPC3) (at 1E14 vg·kg⁻¹), orvehicle, HBSS.

FIG. 13B is a plot showing left ventricular mass normalized to bodyweight (LVM/BW) progression in homozygous Mybpc^(−/−) mice that wereinjected retro-orbitally at two weeks of age with AAV9 encoding themouse Mybpc3 gene (mMybpc3) (at 1E14 vg·kg⁻¹), AAV9 encoding the humanMWYBPC3 gene (hMYBPC3) (at 1E14 vg·kg⁻¹), or vehicle, HBSS.

FIG. 13C is a bar graph left ventricular posterior wall thickness duringdiastole (LVPW;d) in homozygous Mybpc3^(−/−) mice that were injectedretro-orbitally at two weeks of age with AAV9 encoding the mouse Mybpc3gene (mMybpc3) (at 1E14 vg·kg⁻¹), AAV9 encoding the human MYBPC3 gene(hMYBPC3) (at 1E14 vg·kg⁻¹), or vehicle, HBSS.

FIG. 14A is a bar graph showing ejection fraction in homozygous Mybpc3′¹mice that were injected retro-orbitally at two weeks of age with 1E13vg·kg⁻¹, 1E14 vg·kg⁻¹, and 3E14 vg·kg⁻¹ of test vector encoding thehuman MYBPC3 gene or vehicle, HBSS.

FIG. 14B is a bar graph showing ejection fraction compared to pre-dosebaseline (AEF) in homozygous Mybpc3^(−/−) mice that were injectedretro-orbitally at two weeks of age with 1E13 vg·kg⁻¹, 1E14 vg·kg⁻¹, and3E14 vg·kg⁻¹ of test vector encoding the human MYBPC3 gene or vehicle,HBSS.

FIG. 14C is a bar graph showing left ventricular mass normalized to bodyweight (LVM/BW) in homozygous Mybpc^(−/−) mice that were injectedretro-orbitally at two weeks of age with 1E13 vg·kg⁻¹, 1E14 vg·kg⁻¹, and3E14 vg·kg⁻¹ of test vector encoding the human MYBPC3 gene or vehicle,HBSS.

DETAILED DESCRIPTION

The present disclosure provides compositions and methods for genetherapy in cardiac cells and/or with large genes. The disclosedpolynucleotides and vectors may be used in treatment or prevention ofdisease (e.g. heart disease, such as cardiomyopathy). The presentdisclosure provides cardiac-specific promoters, expression cassettes,recombinant adeno-associated virus (rAAV) viral genomes, rAAV virions,pharmaceutical compositions, and methods of use. The expressioncassettes and rAAV viral genomes may comprise a cardiac-specificpromoter operably linked to a polynucleotide encoding a gene product.The gene product may be a therapeutic gene product, such therapeuticgene product used to treat and/or prevent heart disease, e.g.cardiomyopathy. The disclosure further provides rAAV viral genomes andexpression cassettes engineered to deliver and express a large geneproduct. In some embodiments, the vector genome comprises apolynucleotide encoding a MYBPC3 polypeptide, or a functional variantthereof and a promoter. In some embodiments, the promoter is a cardiactroponin T promoter (i.e., a TNNT2 promoter). In some embodiments, therAAV vector genome comprises an expression cassette comprising apolynucleotide encoding a gene product, e.g. MYBPC3 and a promoter, e.g.TNNT2 promoter, flanked by one or more inverted terminal repeatpolynucleotide sequences. In some embodiments, the rAAV virion comprisesa polynucleotide comprising an rAAV vector genome as described hereinand an AAV capsid protein. The present disclosure also providespharmaceutical compositions comprising the vector genomes, rAAV vectorgenomes, and rAAV virions described herein. Also provided are methods oftreating and/or preventing a cardiomyopathy in a subject comprisingadministering the rAAV virions or vector genomes described herein.

Other embodiments, features, and advantages of the invention will beapparent from and encompassed by the following detailed description andclaims.

I. Definitions

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural references unless the contentclearly dictates otherwise.

As used in this specification, the term “and/or” is used in thisdisclosure to mean either “and” or “or” unless indicated otherwise.

Throughout this specification, unless the context requires otherwise,the words “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element or integeror group of elements or integers but not the exclusion of any otherelement or integer or group of elements or integers.

As used in this application, the terms “about” and “approximately” areused as equivalents. Any numerals used in this application with orwithout about/approximately are meant to cover any normal fluctuationsappreciated by one of ordinary skill in the relevant art. In certainembodiments, the term “approximately” or “about” refers to a range ofvalues that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%,12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in eitherdirection (greater than or less than) of the stated reference valueunless otherwise stated or otherwise evident from the context (exceptwhere such number would exceed 100% of a possible value).

The terms “polynucleotide” and “nucleic acid,” used interchangeablyherein, refer to a polymeric form of nucleotides of more than about 100nucleotides, either ribonucleotides or deoxyribonucleotides. Thus, thisterm includes, but is not limited to, single-, double-, ormulti-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or apolymer comprising purine and pyrimidine bases or other natural,chemically or biochemically modified, non-natural, or derivatizednucleotide bases. “Oligonucleotide” generally refers to polynucleotidesof between about 5 and about 100 nucleotides of single- ordouble-stranded DNA. However, for the purposes of this disclosure, thereis no upper limit to the length of an oligonucleotide. Oligonucleotidesare also known as “oligomers” or “oligos” and may be isolated fromgenes, or chemically synthesized by methods known in the art. The terms“polynucleotide” and “nucleic acid” should be understood to include, asapplicable to the embodiments being described, single-stranded (such assense or antisense) and double-stranded polynucleotides.

The term “promoter” as used herein refers a polynucleotide sequence thathas one or more recognition site(s) to which an RNA polymerase binds,such that in a host or target cell, an RNA polymerase may initiate andtranscribe a polynucleotide sequence “downstream” of the promoter intoan RNA. Similarly stated, a “promoter” is operably linked or operativelylinked to a polynucleotide sequence if in a host or target cell in whichthe promoter is active, an RNA polymerase initiates transcription of thepolynucleotide at a transcription state site. Promoters operative inmammalian cells generally comprise an AT-rich region locatedapproximately 25 to 30 bases upstream from the site where transcriptionis initiated and/or another sequence found 70 to 80 bases upstream fromthe start of transcription, a CNCAAT region where N may be anynucleotide.

The terms “upstream” and “upstream end” refer to a portion of apolynucleotide that is, with reference to a transcription start site(TSS), 5′ to the TSS on the sense strand (or coding strand) of thepolynucleotide; and 3′ to the TSS on the antisense strand of thepolynucleotide. The terms “downstream” and “downstream end” refer to aportion of a polynucleotide that is, with reference to a TSS, 3′ to TSSon the sense strand (or coding strand) of the polynucleotide; and 5′ tothe TSS on the antisense strand of the polynucleotide. Thus, a deletionfrom the upstream end of a promoter is a deletion of one or more basepairs in the non-transcribed region of the polynucleotide, 5′ to the TSSon the sense strand (or equivalently, 3′ to the TSS on the antisensestrand). A deletion from the downstream end of a promoter is a deletionof one or more base pairs in the transcribed region of thepolynucleotide, 3′ to the TSS on the sense strand (or equivalently, 5′to the TSS on the antisense strand).

As used herein, the term “transgene” refers to a nucleic acid sequenceencoding a protein or RNA (e.g., a therapeutic protein), which is partlyor entirely heterologous, i.e., foreign, to the transgenic animal orcell into which it is introduced, or, is homologous to an endogenousgene of the transgenic animal or cell into which it is introduced, butwhich is designed to be inserted, or is inserted, into the animal'sgenome in such a way as to alter the genome of the cell into which it isinserted (e.g., it is inserted at a location which differs from that ofthe natural gene or its insertion results in a knockout). A transgenecan include one or more transcriptional regulatory sequences and anyother nucleic acid, such as introns, that may be necessary for optimalexpression of a selected nucleic acid.

The term “sequence identity” refers to the percentage of bases or aminoacids between two polynucleotide or polypeptide sequences that are thesame, and in the same relative position. As such one polynucleotide orpolypeptide sequence has a certain percentage of sequence identitycompared to another polynucleotide or polypeptide sequence. For sequencecomparison, typically one sequence acts as a reference sequence, towhich test sequences are compared. The term “reference sequence” refersto a molecule to which a test sequence is compared.

Methods of sequence alignment for comparison and determination ofpercent sequence identity is well known in the art. Optimal alignment ofsequences for comparison can be conducted, e.g., by the homologyalignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443(1970), by the search for similarity method of Pearson and Lipman, Proc.Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, 575 Science Dr.,Madison, Wis.), by manual alignment and visual inspection (see, e.g.,Brent et al., Current Protocols in Molecular Biology (2003)), by use ofalgorithms know in the art including the BLAST and BLAST 2.0 algorithms,which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402(1977); and Altschul et al., J. Mol. Biol. 215:403-410 (1990),respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information.

In some embodiments, the determination of the percentage of sequenceidentity may take place after a local alignment. Such alignments arewell known in the art, for instance the service EMBOSS Matcheridentifies local similarities between two sequences using an algorithmbased on the LALIGN application, version 2.0u4. In an example, theidentity between two nucleic acid sequences may be calculated using theservice Matcher (EMBOSS) set to the default parameters, e.g. matrix(DNAfull), gap open (16), gap extend (4), alternative matches (1).

An “expression cassette” or “expression construct” refers to a DNApolynucleotide sequence operably linked to a promoter. “Operably linked”or “operatively linked” refers to a juxtaposition wherein the componentsso described are in a relationship permitting them to function in theirintended manner. For instance, a promoter is operably linked to apolynucleotide sequence if the promoter affects the transcription orexpression of the polynucleotide sequence.

As used herein, the term “delivery”, which is used interchangeably with“transduction,” refers to the process by which exogenous nucleic acidmolecules are transferred into a cell such that they are located insidethe cell. Delivery of nucleic acids is a distinct process fromexpression of nucleic acids.

The term “modified” refers to a substance or compound (e.g., a cell, apolynucleotide sequence, and/or a polypeptide sequence) that has beenaltered or changed as compared to the corresponding unmodified substanceor compound.

The term “sample” refers to a biological composition (e.g., a cell or aportion of a tissue) that is subjected to analysis and/or geneticmodification. In some embodiments, a sample is a “primary sample” inthat it is obtained directly from a subject; in some embodiments, a“sample” is the result of processing of a primary sample, for example toremove certain components and/or to isolate or purify certain componentsof interest.

The term “gene” or “recombinant gene” refers to a nucleic acidcomprising an open reading frame encoding a polypeptide, including bothexon and (optionally) intron sequences.

The term “transfection” refers to the uptake of foreign DNA by a cell. Acell has been “transfected” when exogenous DNA has been introducedinside the cell membrane. A number of transfection techniques aregenerally known in the art. See, e.g., Graham et al., Virology 52:456(1973); Sambrook et al., Molecular Cloning: A Laboratory Manual (1989);Davis et al., Basic Methods in Molecular Biology (1986); Chu et al.,Gene 13:197 (1981). Such techniques can be used to introduce one or moreexogenous DNA moieties, such as a nucleotide integration vector andother nucleic acid molecules, into suitable host cells. The termcaptures chemical and electrical transfection procedures.

The term “expression” refers to the process by which a nucleic acid istranslated into peptides or is transcribed into RNA, which, for example,can be translated into peptides, polypeptides or proteins. If thenucleic acid is derived from genomic DNA, expression may, if anappropriate eukaryotic host cell or organism is selected, includesplicing of the mRNA. For heterologous nucleic acid to be expressed in ahost cell, it must initially be delivered into the cell and then, oncein the cell, ultimately reside in the nucleus.

The term “gene therapy” involves the transfer of heterologous DNA tocells of a mammal, particularly a human, with a disorder or conditionsfor which therapy or diagnosis is sought. The DNA is introduced into theselected target cells in a manner such that the heterologous DNA isexpressed and a therapeutic product encoded thereby is produced.Alternatively, the heterologous DNA may in some manner mediateexpression of DNA that encodes the therapeutic product; it may encode aproduct, such as a peptide or RNA that in some manner mediates, directlyor indirectly, expression of a therapeutic product. Gene therapy mayalso be used to deliver nucleic acid encoding a gene product to replacea defective gene or supplement a gene product produced by the mammal orthe cell in which it is introduced. The introduced nucleic acid mayencode a therapeutic gene product that is not normally produced in themammalian host or that is not produced in therapeutically effectiveamounts or at a therapeutically useful time. The heterologous DNAencoding the therapeutic product may be modified prior to introductioninto the cells of the afflicted host in order to enhance or otherwisealter the product or expression thereof.

As used herein, a “heterologous” polynucleotide or nucleic acid refersto a polynucleotide or portion of a polynucleotide derived from a sourceother than the host organism or, for a viral vector, the native,non-recombinant virus. Examples of heterologous DNA include, but are notlimited to, DNA that encodes traceable marker proteins, such as aprotein that confers drug resistance, DNA that encodes therapeuticallyeffective substances, such as anti-cancer agents, enzymes and hormones,and DNA that encodes other types of proteins, such as antibodies.

The term “wild type” refers to the naturally-occurring polynucleotidesequence encoding a protein, or a portion thereof, or protein sequence,or portion thereof, respectively, as it normally exists in vivo in anormal or healthy subject.

The term “variant” refers to a protein or nucleic acid having one ormore genetic changes (e.g. insertions, deletions, substitutions, or thelike) that returns all or substantially all of the functions of thereference protein or nucleic acid. For example, a variant of atherapeutic protein retains the same or substantially the same activityand/or provides the same or substantially the same therapeutic benefitto a subject in need thereof. A variant of a promoter sequence retainsthe ability to initiate transcription at the same or substantially thesame level as the reference promoter, and retains the same orsubstantially the same cell type specificity. In particular embodiments,polynucleotides variants have at least or about 50%, 55%, 60%, 65%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% sequence identity to a reference sequence. In particularembodiments, protein variants have at least or about 50%, 55%, 60%, 65%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100% sequence identity to a reference sequence.

The term “subject” includes animals, such as e.g. mammals. In someembodiments, the mammal is a primate. In some embodiments, the mammal isa human. In some embodiments, subjects are livestock such as cattle,sheep, goats, cows, swine, and the like; or domesticated animals such asdogs and cats. In some embodiments (e.g., particularly in researchcontexts) subjects are rodents (e.g., mice, rats, hamsters), rabbits,primates, or swine such as inbred pigs and the like. The terms “subject”and “patient” are used interchangeably herein.

The term “administering” to a subject is a procedure by which one ormore delivery agents, together or separately, are introduced into orapplied onto a subject such that target cells which are present in thesubject are eventually contacted with the agent.

“Treating” as used herein refers to delivering an agent or compositionto a subject to affect a physiologic outcome.

As used herein, the term “gene product” refers to the a protein ornucleic acid produced by the transcription of a polynucleotide and, inthe case of a protein gene product, the subsequent translation oftranscript into a protein. A “therapeutic gene product” refers to a geneproduct that provides a therapeutic physiological effect or benefit to asubject in need when expressed in a therapeutic amount in a subject.

As used herein, the term “therapeutic protein” refers to a protein orpolypeptide that provides a therapeutic physiological effect or benefitto a subject in need when expressed or administered in a therapeuticamount in a subject. In some embodiments, treatment with a therapeuticprotein or a vector that expresses a therapeutic protein provides atherapeutic physiological effect or benefit to a subject with heartdisease (e.g., a subject with cardiomyopathy). Illustrative therapeuticproteins for the treatment of heart disease are provided in Table 2.

As used herein, the term “cardiomyopathy” refers to the deterioration ofthe function of the myocardium (i.e., the actual heart muscle) for anyreason. Subjects with cardiomyopathy are often at risk of arrhythmia orsudden cardiac death or both.

As used herein, the term “hypertrophic cardiomyopathy” refers to adisease of the heart and myocardium in which a portion of the myocardiumis hypertrophied.

As used herein, the term “familial hypertrophic cardiomyopathy” refersto a genetic disorder characterized by increased growth (i.e.,hypertrophy) in thickness of the wall of the left ventricle.

As used herein, the term “effective amount” refers to the minimum amountof an agent or composition required to result in a particularphysiological effect. The effective amount of a particular agent may berepresented in a variety of ways based on the nature of the agent, suchas mass/volume, # of cells/volume, particles/volume, (mass of theagent)/(mass of the subject), # of cells/(mass of subject), orparticles/(mass of subject). The effective amount of a particular agentmay also be expressed as the half-maximal effective concentration(EC₅₀), which refers to the concentration of an agent that results in amagnitude of a particular physiological response that is half-waybetween a reference level and a maximum response level.

II. Polynucleotides

In some embodiments, the present disclosure provides polynucleotidesequences for the treatment and/or prevention of heart disease (e.g.,cardiomyopathy). In some embodiments, the polynucleotide sequencescomprise a cardiac-specific promoter operatively linked to apolynucleotide encoding one or more therapeutic gene products for thetreatment and/or prevention of cardiomyopathy.

Polynucleotides refer to a polymeric form of nucleotides of at least 5,at least 10, at least 15, at least 20, at least 25, at least 30, atleast 40, at least 50, at least 100, at least 200, at least 300, atleast 400, at least 500, at least 1000, at least 5000, at least 10000,or at least 15000 or more nucleotides in length, either ribonucleotidesor deoxyribonucleotides or a modified form of either type of nucleotide,as well as all intermediate lengths. “Intermediate lengths,” in thiscontext, means any length between the quoted values, such as 6, 7, 8, 9,etc., 101, 102, 103, etc.; 151, 152, 153, etc.; 201, 202, 203, etc.

As a result of the degeneracy of the genetic code, there are manynucleotide sequences that encode a polypeptide, or fragment of variantthereof, as described herein. Some of these polynucleotides bear minimalhomology to the nucleotide sequence of any native gene. Nonetheless,polynucleotides that vary due to differences in codon usage arespecifically contemplated in particular embodiments, for examplepolynucleotides that are optimized for human and/or primate codonselection. Further, alleles of the genes comprising the polynucleotidesequences provided herein may also be used. Alleles are endogenous genesthat are altered as a result of one or more mutations, such asdeletions, additions and/or substitutions of nucleotides.

The polynucleotides contemplated herein, regardless of the length of thecoding sequence itself, may be combined with other DNA sequences, suchas promoters and/or enhancers, untranslated regions (UTRs), signalsequences, Kozak sequences, polyadenylation signals, additionalrestriction enzyme sites, multiple cloning sites, internal ribosomalentry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, andAtt sites), termination codons, transcriptional termination signals, andpolynucleotides encoding self-cleaving polypeptides, epitope tags, asdisclosed elsewhere herein or as known in the art.

Polynucleotides can be prepared, manipulated and/or expressed using anyof a variety of well-established techniques known and available in theart.

In some embodiments, the polynucleotide sequence is a promoter. In someembodiments, the polynucleotide sequence is a promoter operativelylinked to a polynucleotide encoding a therapeutic gene product for thetreatment or prevention of heart disease (e.g., cardiomyopathy).

In some embodiments, the vector comprises a cardiac-specific promoterwhich is operably linked to a polynucleotide encoding a therapeutic geneproduct (e.g., encoding a therapeutic protein, e.g., MYBPC3 protein). Asused herein, a “cardiac-specific promoter” refers to a promoter whoseactivity in cardiac cells is at least 2-fold higher than in any othernon-cardiac cell type. Preferably, a cardiac-specific promoter suitablefor being used in the vector of the invention has an activity in cardiaccells which is at least 5-fold, at least 10-fold, at least 15-fold, atleast 20-fold, at least 25-fold, or at least 50-fold higher compared toits activity in a non-cardiac cell type.

In some embodiments, the vector comprises a cardiomyocyte-specificpromoter which is operably linked to a polynucleotide encoding atherapeutic gene product (e.g., MYBPC3 protein). A“cardiomyocyte-specific promoter”, as used herein, specifies a promoterwhose activity in cardiomyocytes is at least 2-fold higher than in anyother non-cardiac cell type or cardiac cell which is not acardiomyocyte. Preferably, a cardiomyocyte-specific promoter suitablefor being used in the vector of the present disclosure has an activityin cardiomyocytes which is at least 5-fold, at least 10-fold, at least15-fold, at least 20-fold, at least 25-fold, or at least 50-fold highercompared to its activity in a non-cardiac cell type or a cardiac celltype which is not a cardiomyocyte.

In some embodiments, the cardiac-specific or cardiomyocyte-specificpromoter is a human promoter. Examples of cardiac-specific orcardiomyocyte-specific promoter include, but are not limited to, thealpha myosin heavy chain promoter, the myosin light chain 2v promoter,the alpha myosin heavy chain promoter, the alpha-cardiac actin promoter,the alpha-tropomyosin promoter, the cardiac troponin C promoter, thecardiac troponin I promoter, the cardiac myosin-binding protein Cpromoter, and the sarco/endoplasmic reticulum Ca²⁺ ATPase (SERCA)promoter (e.g. isoform 2 of SERCA2).

In some embodiments, the cardiac-specific promoter is the cardiac TNNT2promoter. In some embodiments, the cardiac TNNT2 promoter is modified,e.g., by the deletion, insertion, or substitution of polynucleotides.Illustrative polynucleotide sequences of the cardiac TNNT2 promoter areshown in Table 1 below. The transcription start site (TSS) of the TNNT2promoters are bolded and underlined.

TABLE 1 Illustrative TNNT2 promoters SEQ ID Name DNA Sequence NO.TNNT2p-600 GTCATGGACAAGACCCACCT 1 TGCAGATGTCCTCACTGGGGCTGGCAGAGCCGGCAACCTG CCTAAGGCTGCTCAGTCCAT TAGGAGCCAGTAGCCTGGAAGATGTCTTTACCCCCAGCAT CAGTTCAAGTGGAGCAGCAC ATAACTCTTGCCCTCTGCCTTCCAAGATTCTGGTGCTGAG ACTTATGGAGTGTCTTGGAG GTTGCCTTCTGCCCCCCAACCCTGCTCCCAGCTGGCCCTC CCAGGCCTGGGTTGCTGGCC TCTGCTTTATCAGGATTCTCAAGAGGGACAGCTGGTTTAT GTTGCATGACTGTTCCCTGC ATATCTGCTCTGGTTTTAAATAGCTTATCTGAGCAGCTGG AGGACCACATGGGCTTATAT GGCGTGGGGTACATGTTCCTGTAGCCTTGTCCCTGGCACC TGCCAAAATAGCAGCCAACA CCCCCCACCCCCACCGCCATCCCCCTGCCCCACCCGTCCC CTGTCGCACATTCCTCCCTC CGCAGGGCTGGCTCACCAGGCCCCAGCCCACATGCCTGCT TAAAGCCCT C TCCATCCTCT GCCTCACCCAGTCCCCGCTGAGACTGAGCAGACGCCTCCA TNNT2p-500 GATGTCTTTACCCCCAGCAT 2CAGTTCAAGTGGAGCAGCAC ATAACTCTTGCCCTCTGCCT TCCAAGATTCTGGTGCTGAGACTTATGGAGTGTCTTGGAG GTTGCCTTCTGCCCCCCAAC CCTGCTCCCAGCTGGCCCTCCCAGGCCTGGGTTGCTGGCC TCTGCTTTATCAGGATTCTC AAGAGGGACAGCTGGTTTATGTTGCATGACTGTTCCCTGG ATATCTGCTCTGGTTTTAAA TAGCTTATCTGAGCAGCTGGAGGACCACATGGGCTTATAT GGCGTGGGGTACATGTTCCT GTAGCCTTGTCCCTGGCACCTGCCAAAATAGCAGCCAACA CCCCCCACCCCCACCGCCAT CCCCCTGCCCCACCCGTCCCCTGTCGCACATTCCTCCCTC CGCAGGGCTGGCTCACCAGG CCCCAGCCCACATGCCTGCT TAAAGCCCTC TCCATCCTCT GCCTCACCCAGTCCCCGCTG AGACTGAGCAGACGCCTCCA TNNT2p-400GTTGCCTTCTGCCCCCCAAC 3 CCTGCTCCCAGCTGGCCCTC CCAGGCCTGGGTTGCTGGCCTCTGCTTTATCAGGATTCTC AAGAGGGACAGCTGGTTTAT GTTGCATGACTGTTCCCTGCATATCTGCTCTGGTTTTAAA TAGCTTATCTGAGCAGCTGG AGGACCACATGGGCTTATATGGCGTGGGGTACATGTTCCT GTAGCCTTGTCCCTGGCACC TGCCAAAATAGCAGCCAACACCCCCCACCCCCACCGCCAT CCCCCTGCCCCACCCGTCCC CTGTCGCACATTCCTCCCTCCGCAGGGCTGGCTCACCAGG CCCCAGCCCACATGCCTGCT TAAAGCCCT C TCCATCCTCTGCCTCACCCAGTCCCCGCTG AGACTGAGCAGACGCCTCCA TNNT2p-300GTTGCATGACTGTTCCCTGC 4 ATATCTGCTCTGGTTTTAAA TAGCTTATCTGAGCAGCTGGAGGACCACATGGGCTTATAT GGCGTGGGGTACATGTTCCT GTAGCCTTGTCCCTGGCACCTGCCAAAATAGCAGCCAACA CCCCCCACCCCCACCGCCAT CCCCCTGCCCCACCCGTCCCCTGTCGCACATTCCTCCCTC CGCAGGGCTGGCTCACCAGG CCCCAGCCCACATGCCTGCT TAAAGCCCTC TCCATCCTCT GCCTCACCCAGTCCCCGCTG AGACTGAGCAGACGCCTCCA

In some embodiments, the cardiac TNNT2 promoter is modified to comprisea polynucleotide sequence of between about 200 and 500 base pairs,between about 250 and 500 base pairs, between about 300 to 500 basepairs, between about 350 to 500 base pairs, between about 400 to 500base pairs, between about 450 to 500 base pairs, between about 200 and450 base pairs, between about 200 and 400 base pairs, between about 200and 350 base pairs, between about 200 and 300 base pairs, and betweenabout 200 and 250 base pairs in length. In some embodiments, themodified cardiac TNNT2 promoter comprises a polynucleotide sequence ofbetween about 350 base pairs to about 450 base pairs, between about 375base pairs to about 425 base pairs, between about 375 base pairs toabout 400 base pairs, between about 375 base pairs to about 425 basepairs, between about 400 base pairs to about 425 base pairs, or betweenabout 400 base pairs to about 450 base pairs. In some embodiments, thecardiac TNNT2 promoter comprises a polynucleotide sequence of about 400base pairs.

In a particular embodiment, the modified cardiac troponin T promotercomprises between 300 bp and 500 bp of SEQ ID NO: 1. For instance, themodified cardiac troponin T promoter may comprise SEQ ID NO: 3. In someexamples, the 300 bp-500 bp sequence may be linked to furtherpolynucleotide sequences but may not be linked to additional sequencesderived from SEQ ID NO: 1. For example, in an embodiment, the modifiedcardiac troponin T promoter may include no more than 500 bp of SEQ IDNO: 1 but may include additional unrelated polynucleotide sequences. Inanother example, the modified cardiac troponin T promoter may includeSEQ ID NO: 3, no additional sequences derived from SEQ ID NO: 1, but mayinclude additional unrelated polynucleotide sequences.

In some embodiments, the cardiac TNNT2 promoter is modified by thedeletion of polynucleotides. A modification may include one, two, threeor more internal deletions. Each deletion may be a deletion of 1 basepair, 2 base pairs, 3 base pairs, 4 base pairs, 5 base pairs, 10 basepairs, 15 base pairs, 20 base pairs, 25 base pairs, 30 base pairs, 40base pairs, 50 base pairs, 60 base pairs, 70 base pairs, 80 base pairs,90 base pairs, 100 base pairs, 125 base pairs, 150 base pairs, 175 basepairs, 200 base pairs, 225 base pairs, 250 base pairs, 275 base pairs,or 300 base pairs with respect to a reference cardiac TNNT2 promoter(SEQ ID NO: 1) having about 600 base pairs.

In some embodiments, the TNNT2 promoter is modified by the deletion ofpolynucleotides from the upstream end of the promoter with respect to areference cardiac TNNT2 promoter (SEQ ID NO: 1) having about 600 basepairs. A modification may include the deletion of 1 base pair, 2 basepairs, 3 base pairs, 4 base pairs, 5 base pairs, 10 base pairs, 15 basepairs, 20 base pairs, 25 base pairs, 30 base pairs, 40 base pairs, 50base pairs, 60 base pairs, 70 base pairs, 80 base pairs, 90 base pairs,100 base pairs, 125 base pairs, 150 base pairs, 175 base pairs, 200 basepairs, 225 base pairs, 250 base pairs, 275 base pairs, or 300 base pairsfrom the upstream end of the promoter with respect to a referencecardiac TNNT2 promoter (SEQ ID NO: 1) having about 600 base pairs. Insome embodiments, the modification is a 200 base pair deletion from theupstream end of the promoter with respect to a reference cardiac TNNT2promoter (SEQ ID NO: 1) having about 600 base pairs.

In some embodiments, the cardiac TNNT2 promoter is modified by thedeletion of polynucleotides from the downstream end of the promoter withrespect to a reference cardiac TNNT2 promoter (SEQ ID NO: 1) havingabout 600 base pairs. A modification may include the deletion of 1 basepair, 2 base pairs, 3 base pairs, 4 base pairs, 5 base pairs, 10 basepairs, 15 base pairs, 20 base pairs, 25 base pairs, 30 base pairs, 40base pairs, 50 base pairs, 60 base pairs, 70 base pairs, 80 base pairs,90 base pairs, 100 base pairs, 125 base pairs, 150 base pairs, 175 basepairs, 200 base pairs, 225 base pairs, 250 base pairs, 275 base pairs,or 300 base pairs from the downstream end of the promoter with respectto a reference cardiac TNNT2 promoter (SEQ ID NO: 1) having about 600base pairs.

In some embodiments, the cardiac TNNT2 promoter is modified by aninternal deletion of polynucleotides. A modification may include theinternal deletion of 1 base pair, 2 base pairs, 3 base pairs, 4 basepairs, 5 base pairs, 10 base pairs, 15 base pairs, 20 base pairs, 30base pairs, 40 base pairs, 50 base pairs, 60 base pairs, 70 base pairs,80 base pairs, 90 base pairs, 100 base pairs, 125 base pairs, 150 basepairs, 175 base pairs, 200 base pairs, 225 base pairs, 250 base pairs,275 base pairs, or 300 base pairs with respect to a reference cardiacTNNT2 promoter (SEQ ID NO: 1).

In some embodiments, the cardiac TNNT2 promoter is modified by theinsertion of polynucleotides. A modification may include the insertionof 1 base pair, 2 base pairs, 3 base pairs, 4 base pairs, 5 base pairs,10 base pairs, 15 base pairs, 20 base pairs, 25 base pairs, 30 basepairs, 35 base pairs, 40 base pairs, 45 base pairs, 50 base pairs, 55base pairs, 60 base pairs, 65 base pairs, 70 base pairs, 75 base pairs,80, base pairs, 85 base pairs, 90 base pairs, 100 base pairs, 125 basepairs, 150 base pairs, 175 base pairs, 200 base pairs, 225 base pairs,250 base pairs, 275 base pairs, or 300 base pairs with respect to areference cardiac TNNT2 promoter (SEQ ID NO: 1).

In some embodiments, the cardiac TNNT2 promoter is modified by thesubstitution of polynucleotides. A modification may include thesubstitution of 1 base pair, 2 base pairs, 3 base pairs, 4 base pairs, 5base pairs, 6 base pairs, 7 base pairs, 8 base pairs, 9 base pairs, or10 base pairs with respect to a reference cardiac TNNT2 promoter (SEQ IDNO: 1).

In some embodiments, the polynucleotide sequence of the TNNT2 promotershares at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity with the polynucleotide sequence −450 base pairs to +1 basepairs relative to the transcription start site of the human TNNT2 gene.In some embodiments, the polynucleotide sequence of the TNNT2 promotershares at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or and 100% sequenceidentity with the polynucleotide sequence −350 base pairs to +1 basepairs relative to the transcription start site of the human TNNT2 gene.In some embodiments, the polynucleotide sequence of the TNNT2 promotershares at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or and 100% sequenceidentity with the polynucleotide sequence −250 base pairs to +1 basepairs relative to the transcription start site of the human TNNT2 gene.

In some embodiments, the polynucleotide sequence of the cardiac TNNT2promoter shares at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or and 100%sequence identity with the polynucleotide sequence −450 base pairs to+50 base pairs relative to the transcription start site of the TNNT2gene. In some embodiments, the polynucleotide sequence of the cardiacTNNT2 promoter shares at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or and100% sequence identity with the polynucleotide sequence −350 base pairsto +50 base pairs relative to the transcription start site of the TNNT2gene. In some embodiments, the polynucleotide sequence of the cardiacTNNT2 promoter shares at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or and100% sequence identity with the polynucleotide sequence −250 base pairsto +5 base pairs relative to the transcription start site of the TNNT2gene.

In some embodiments, the cardiac TNNT2 promoter comprises apolynucleotide comprising a sequence that shares at least 80%, 90%, 95%,96%, 97%, 98%, 99%, or and 100% identity to any one of SEQ ID NOs: 1-85.In some embodiments, the polynucleotide comprises a sequence that sharesat least 80% a identity to any one of SEQ ID NOS: 1-85. In someembodiments, the polynucleotide comprises a sequence that shares atleast 90% identity to any one of SEQ ID NOS: 1-85. In some embodiments,the polynucleotide comprises a sequence that shares at least 100%identity to any one of SEQ ID NOS: 1-85. In some embodiments, thepolynucleotide comprises a sequence that shares at least 80% identity toSEQ ID NO: 1. In some embodiments, the polynucleotide comprises asequence that shares at least 90% identity to SEQ ID NO: 1. In someembodiments, the polynucleotide comprises a sequence that shares atleast 100% identity to SEQ ID NO: 1. In some embodiments, thepolynucleotide comprises a sequence that shares at least 80% identity toSEQ ID NO: 3. In some embodiments, the polynucleotide comprises asequence that shares at least 90% identity to SEQ ID NO: 3. In someembodiments, the polynucleotide comprises a sequence that shares atleast 100% identity to SEQ ID NO: 3.

B. Illustrative Gene Products (Proteins)

The promoters of the disclosure may be operatively linked to apolynucleotide comprising a sequence encoding a gene product (e.g.,protein or nucleic acid). In some embodiments, the gene product is atherapeutic protein. The therapeutic protein may be any of the nativehuman proteins listed in Table 2, or functional homologs or variantsthereof. The promoters of the disclosure are particularly suited for usewith large genes that may otherwise be expressed at low levels or not beexpressed when delivered by a viral vector. An advantage of someembodiments disclosed herein lies in the ability to express atherapeutic protein (particularly a large therapeutic protein) in aviral vector having limited packaging capacity, e.g., an AAV vector. A“large” protein is any protein whose size impacts expression in aselected vector. Generally, “large” therapeutic proteins comprise atleast about 1000 or more amino acids—that is, the protein is encoded bya polynucleotide sequence of about 3 kbps or greater. Illustrativeproteins, including large proteins, are provided in Table 2 below.

TABLE 2 Illustrative Proteins NCBI UniProt Gene Name Gene Symbol Gene IDID Myosin-binding protein C MYBPC3 4607 Q14896 Potassium voltage-gatedchannel KCNH2 3757 Q12809 subfamily H member 2 Transient receptorpotential cation TRPM4 54795 Q8TD43 channel subfamily M member 4 DSG21829 Q14126 Desmoglein-2 ATPase sarcoplasmic/endoplasmic ATP2A2 488P16615 reticulum calcium transporting 2 Calcium voltage-gated channelCACNA1C 775 Q13936 subunit alpha 1C Dystrophin DMD 1756 P11532 DM1protein kinase DMPK 1760 Q09013 Ectopic P granules protein 5 EPG5 57724Q9HCE0 homolog EvC ciliary complex subunit 1 EVC 2121 P57679 Limbin EVC2132884 Q86UK5 Fibrillin-1 FBN1 2200 P35555 Neurofibromin NF1 4763 P21359Sodium channel protein type 5 SCN5A 6331 Q14524 subunit alpha Son ofsevenless homolog 1 SOS1 6654 Q07889 Natriuretic peptide receptor 1 NPR14881 P16066 Receptor tyrosine-protein kinase ERBB4 2066 Q15303 erbB-4Vasoactive intestinal peptide VIP 7432 P01282 Beta-myosin heavy chainMYH7 4625 P12883

Various therapeutic polynucleotides, or therapeutic proteins encoded bypolynucleotides, having lengths of 3 kilobases or greater are expressedmore effectively when operatively linked to a modified TNNT2 promoter ofthe disclosure compared to a TNNT2 promoter of about 600 base pairs. Thepromoters of the disclosure are useful in expression of, at least, thefollowing: a) large genes in which loss-of-function mutations result incardiomyopathy (gene replacement therapy); b) large genes whoseexpression in cardiomyocytes is cardioprotective; c) combinations ofgenes whose co-expression in cardiomyocytes is beneficial; and d) toolsfor cardiomyocyte-specific genome editing. A “large” gene is any genewhose size impacts expression in a selected vector. Generally, “large”therapeutic genes encode proteins that comprise at least about 1000 ormore amino acids—that is, the gene comprises a polynucleotide sequenceof about 3 kbps or greater. In further embodiments, the vectors andpromoters of the disclosure are used in treatment of the diseases ordisorders list in Table 3, where the polynucleotide encodes thetherapeutic protein indicated in the table.

TABLE 3 Illustrative Therapeutic Gene Products for Heart DiseaseTherapeutic Gene Gene Condition Product Size (kb) Timothy syndromeCACNA1C 6.663 Becker muscular dystrophy DMD 11.055 Duchenne musculardystrophy DMD 11.055 Myotonic dystrophy type 1 DMPK 4.653 Vici syndromeEPG5 7.737 Ellis-Van Creveld syndrome EVC 2.976 Ellis-Van Creveldsyndrome EVC2 3.924 Marfan syndrome FBN1 8.613 Long QT Syndrome KCNH23.477 Neurofibromatosis Noonan syndrome NT1 3.517 Brugada Syndrome SCN5A6.048 Long QT Syndrome SCN5A 6.048 Paroxysmal ventricular fibrillation 1SCN5A 6.048 Progressive familial heart block type 1A SCN5A 6.048 Noonansyndrome SOS1 3.999 Progressive familial heart block type 1B TRPM4 3.642Acute decompensated heart failure (ADHF) NPR1 3.183 Congestive HeartFailure (CHF) ERBB4 3.924 Congestive Heart Failure (CHF) VIP analog*3.729 Hypertrophic cardiomyopathy MYBPC3 3.822 Left VentricularNoncompaction MYBPC3 3.822 Cardiomyopathy Hypertrophic cardiomyopathyMYH7 5.805 Left Ventricular Noncompaction MYH7 5.805 Cardiomyopathy *VIPfused to an ELP biopolymer (e.g., PB1046) to increase stability in vivo

MYBPC3 is a gene expressed in cardiac cells. Various mutations in MYBPC3are known to cause hypertrophic cardiomyopathy. Almost half of allmutations causative for hypertrophic cardiomyopathy result intruncations, via nonsense, frameshift or splice-site mutations (Marianand Braunwald, Circ. Res. 121:749-770 (2017); Walsh et al., Genet. Med.19:192-203 (2017). mRNAs containing premature stop codons are subjectedto surveillance and degradation by nonsense-mediated decay machinery.This is consistent with decreased levels of mutant RNAs in analysis ofcardiac tissue from hypertrophic cardiomyopathy patients who havereceived myectomies (Marston et al., Circ. Res. 105:219-222 (2009); vanDijk et al., Circulation 119:1473-1483 (2009), Helms et al., Circ.Cardiovasc. Genet. 7:434-443 (2014). Further, any resultant truncatedpolypeptides appear sensitive to the ubiquitin-proteasome degradationsystem. In patient myectomy samples, no truncated protein was observedfor nine distinct mutations (Rottbauer et al., J. Clin. Invest.100:475-482 (1997); Moolman et al., Circulation 101:1396-1402 (2000);Marston et al., Circ. Res. 105:219-222 (2009); van Dijk et al., Circ.Heart Fail 5:3646 (2012)). Even though it appears that the wild-typeMYBPC3 allele in heterozygous patients is slightly upregulated, thetotal amount of MYBPC3 protein incorporated into sarcomeres fallssignificantly below normal at ˜65% (Marston et al., Circ. Res.105:219-222 (2009); van Dijk et al., Circ. Heart Fail 5:36-46 (2012);McNamara et al., PLoS One 12:e0180064 (2017)). Thus, the sarcomericpathophysiology of hypertrophic cardiomyopathy patients with MWYBPC3truncating mutations appears to be due to haploinsufficiency.

In some embodiments, the disclosure provides a vector comprising apolynucleotide sequence that encodes the MYBPC3 protein, operativelylinked to a modified cardiac TNNT2 promoter. Similarly stated, in someembodiments, the polynucleotide sequence operatively linked to thecardiac-specific promoter (e.g., a modified cardiac TNNT2 promoter) isMYBPC3, or a mutant, variant, or fragment thereof. In humans, the MYBPC3gene encodes the MYBPC3 protein (also known as MyBP-C), which regulatesthe cardiac sarcomere, the basic unit of muscle contraction. The cardiacmuscle sarcomere consists of thick and thin filaments, and MYBPC3attaches to the thick filaments to prevent premature degradation.Illustrative MYBPC3 polynucleotide sequences are shown in Table 4Abelow. In some embodiments, the polynucleotide encoding MYBPC3 shares atleast 85%, 90%, 95%, 99%, or 100% identity to any one of SEQ ID NOs:86-89. Illustrative MYBPC3 protein sequences are shown in Table 4B. Insome embodiments, the vector genome encodes an MYBPC3 protein thatshares at least 85%, 90%, 95%, 99%, or 100% identity to any one of SEQID NOs: 103-106.

TABLE 4A Illustrative MYBPC3 Polynucleotide Sequences SEQ ID NameDNA Sequence NO MYBPC3 ATGCCTGAGCCGGGGAAGAAGCCAG 86TCTCAGCTTTTAGCAAGAAGCCACG GTCAGTGGAAGTGGCCGCAGGCAGCCCTGCCGTGTTCGAGGCCGAGACAG AGCGGGCAGGAGTGAAGGTGCGCTGGCAGCGCGGAGGCAGTGACATCAGC GCCAGCAACAAGTACGGCCTGGCCACAGAGGGCACACGGCATACGCTGAC AGTGCGGGAAGTGGGCCCTGCCGACCAGGGATCTTACGCAGTCATTGCTG GCTCCTCCAAGGTCAAGTTCGACCTCAAGGTCATAGAGGCAGAGAAGGCA GAGCCCATGCTGGCCCCTGCCCCTGCCCCTGCTGAGGCCACTGGAGCCCC TGGAGAAGCCCCGGCCCCAGCCGCTGAGCTGGGAGAAAGTGCCCCAAGTC CCAAAGGGTCAAGCTCAGCAGCTCTCAATGGTCCTACCCCTGGAGCCCCC GATGACCCCATTGGCCTCTTCGTGATGCGGCCACAGGATGGCGAGGTGAC CGTGGGTGGCAGCATCACCTTCTCAGCCCGCGTGGCCGGCGCCAGCCTCC TGAAGCCGCCTGTGGTCAAGTGGTTCAAGGGCAAATGGGTGGACCTGAGC AGCAAGGTGGGCCAGCACCTGCAGCTGCACGACAGCTACGACCGCGCCAG CAAGGTCTATCTGTTCGAGCTGCACATCACCGATGCCCAGCCTGCCTTCA CTGGCAGCTACCGCTGTGAGGTGTCCACCAAGGACAAATTTGACTGCTCC AACTTCAATCTCACTGTCCACGAGGCCATGGGCACCGGAGACCTGGACCT CCTATCAGCCTTCCGCCGCACGAGCCTGGCTGGAGGTGGTCGGCGGATCA GTGATAGCCATGAGGACACTGGGATTCTGGACTTCAGCTCACTGCTGAAA AAGAGAGACAGTTTCCGGACCCCGAGGGACTCGAAGCTGGAGGCACCAGC AGAGGAGGACGTGTGGGAGATCCTACGGCAGGCACCCCCATCTGAGTACG AGCGCATCGCCTTCCAGTACGGCGTCACTGACCTGCGCGGCATGCTAAAG AGGCTCAAGGGCATGAGGCGCGATGAGAAGAAGAGCACAGCCTTTCAGAA GAAGCTGGAGCCGGCCTACCAGGTGAGCAAAGGCCACAAGATCCGGCTGA CCGTGGAACTGGCTGACCATGACGCTGAGGTCAAATGGCTCAAGAATGGC CAGGAGATCCAGATGAGCGGCAGCAAGTACATCTTTGAGTCGATCGGTGC CAAGCGTACCCTGACCATCAGCCAGTGCTCATTGGCGGACGACGCAGCCT ACCAGTGCGTGGTGGGTGGCGAGAAGTGTAGAACGGAGCTCTTTGTGAAA GAGCCCCCTGTGCTCATCACGCGCCCCTTGGAGGACCAGCTGGTGATGGT GGGGCAGCGGGTGGAGTTTGAGTGTGAAGTATCGGAGGAGGGGGCGCAAG TCAAATGGCTGAAGGACGGGGTGGAGCTGACCCGGGAGGAGACCTTCAAA TACCGGTTCAAGAAGGACGGGCAGAGACACCACCTGATCATCAACGAGGC CATGCTGGAGGACGCGGGGCACTATGCACTGTGCACTAGCGGGGGCCAGG CGCTGGCTGAGCTCATTGTGCAGGAAAAGAAGCTGGAGGTGTACCAGAGC ATCGCAGACCTGATGGTGGGCGCAAAGGACCAGGCGGTGTTCAAATGTGA GGTCTCAGATGAGAATGTTCGGGGTGTGTGGCTGAAGAATGGGAAGGAGC TGGTGCCCGACAGCCGCATAAAGGTGTCCCACATCGGGCGGGTCCACAAA CTGACCATTGACGACGTCACACCTGCCGACGAGGCTGACTACAGCTTTGT GCCCGAGGGCTTCGCCTGCAACCTGTCAGCCAAGCTCCACTTCATGGAGG TCAAGATTGACTTCGTACCCAGGCAGGAACCTCCCAAGATCCACCTGGAC TGCCCAGGCCGCATACCAGACACCATTGTGGTTGTAGCTGGAAATAAGCT ACGTCTGGACGTCCCTATCTCTGGGGACCCCGCTCCCACTGTGATCTGGC AGAAGGCTATCACGCAGGGGAATAAGGCCCCAGCCAGGCCAGCCCCAGAT GCCCCAGAGGACACAGGTGACAGCGATGAGTGGGTGTTTGACAAGAAGCT GCTGTGTGAGACCGAGGGCCGGGTCCGCGTGGAGACCACCAAGGACCGCA GCATCTTCACGGTCGAGGGGGCAGAGAAGGAAGATGAGGGCGTCTACACG GTCACAGTGAAGAACCCTGTGGGCGAGGACCAGGTCAACCTCACAGTCAA GGTCATCGACGTGCCAGACGCACCTGCGGCCCCCAAGATCAGCAACGTGG GAGAGGACTCCTGCACAGTACAGTGGGAGCCGCCTGCCTACGATGGCGGG CAGCCCATCCTGGGCTACATCCTGGAGCGCAAGAAGAAGAAGAGCTACCG GTGGATGCGGCTGAACTTCGACCTGATTCAGGAGCTGAGTCATGAAGCGC GGCGCATGATCGAGGGCGTGGTGTACGAGATGCGCGTCTACGCGGTCAAC GCCATCGGCATGTCCAGGCCCAGCCCTGCCTCCCAGCCCTTCATGCCTAT CGGTCCCCCCAGCGAACCCACCCACCTGGCAGTAGAGGACGTCTCTGACA CCACGGTCTCCCTCAAGTGGCGGCCCCCAGAGCGCGTGGGAGCAGGAGGC CTGGATGGCTACAGCGTGGAGTACTGCCCAGAGGGCTGCTCAGAGTGGGT GGCTGCCCTGCAGGGGCTGAGAGAGCACACATCGATACTGGTGAAGGACC TGCCCACGGGGGCCCGGCTGCTTTTCCGAGTGCGGGCACACAATATGGCA GGGCCTGGAGCCCCTGTTACCACCACGGAGCCGGTGACAGTGCAGGAGAT CCTGCAACGGCCACGGCTTCAGCTGCCCAGGCACCTGCGCCAGACCATTC AGAAGAAGGTCGGGGAGCCTGTGAACCTTCTGATCCCTTTCCAGGGCAAG CCCCGGCCTCAGGTGACCTGGACCAAAGAGGGGCAGCCCCTGGCAGGCGA GGAGGTGAGCATCCGCAACAGCCCCACAGACACCATCCTGTTCATCCGGG CCGCTCGCCGCGTGCATTCAGGCACTTACCAGGTGACGGTGCGCATTGAG AACATGGAGGACAAGGCCACGCTGGTGCTGCAGGTTGTTGACAAGCCAAG TCCTCCCCAGGATCTCCGGGTGACTGACGCCTGGGGTCTTAATGTGGCTC TGGAGTGGAAGCCACCCCAGGATGTCGGCAACACGGAACTCTGGGGGTAC ACAGTGCAGAAAGCCGACAAGAAGACCATGGAGTGGTTCACCGTCTTGGA GCATTAGCGCCGCACCCACTGCGTGGTGCCAGAGCTCATCATTGGCAATG GCTACTACTTCCGCGTCTTCAGCCAGAATATGGTTGGCTTTAGTGACAGA GCGGCCACCACCAAGGAGCCCGTCTTTATCCCCAGACCAGGCATCACCTA TGAGCCACCCAACTATAAGGCCCTGGACTTCTCCGAGGCCCCAAGCTTCA CCCAGCCCCTGGTGAACCGCTCGGTCATCGCGGGCTACACTGCTATGCTC TGCTGTGCTGTCCGGGGTAGCCCCAAGCCCAAGATTTCCTGGTTCAAGAA TGGCCTGGACCTGGGAGAAGACGCCCGCTTCCGCATGTTCAGCAAGCAGG GAGTGTTGACTCTGGAGATTAGAAAGCCCTGCCCCTTTGACGGGGGCATC TATGTCTGCAGGGCCACCAACTTACAGGGCGAGGCACGGTGTGAGTGCCG CCTGGAGGTGCGAGTGCCTCAGTAA MYBPC3-ATGCCTGAGCCGGGGAAGAAGCCAG 87 delC3 TCTCAGCTTTTAGCAAGAAGCCACGGTCAGTGGAAGTGGCCGCAGGCAGC CCTGCCGTGTTCGAGGCCGAGACAGAGCGGGCAGGAGTGAAGGTGCGCTG GCAGCGCGGAGGCAGTGACATCAGCGCCAGCAACAAGTACGGCCTGGCCA CAGAGGGCACACGGCATACGCTGACAGTGCGGGAAGTGGGCCCTGCCGAC CAGGGATCTTACGCAGTCATTGCTGGCTCCTCCAAGGTCAAGTTCGACCT CAAGGTCATAGAGGCAGAGAAGGCAGAGCCCATGCTGGCCCCTGCCCCTG CCCCTGCTGAGGCCACTGGAGCCCCTGGAGAAGCCCCGGCCCCAGCCGCT GAGCTGGGAGAAAGTGCCCCAAGTCCCAAAGGGTCAAGCTCAGCAGCTCT CAATGGTCCTACCCCTGGAGCCCCCGATGACCCCATTGGCCTCTTCGTGA TGCGGCCACAGGATGGCGAGGTGACCGTGGGTGGCAGCATCACCTTCTCA GCCCGCGTGGCCGGCGCCAGCCTCCTGAAGCCGCCTGTGGTCAAGTGGTT CAAGGGCAAATGGGTGGACCTGAGCAGCAAGGTGGGCCAGCACCTGCAGC TGCACGACAGCTACGACCGCGCCAGCAAGGTCTATCTGTTCGAGCTGCAC ATCACCGATGCCCAGCCTGCCTTCACTGGCAGCTACCGCTGTGAGGTGTC CACCAAGGACAAATTTGACTGCTCCAACTTCAATCTCACTGTCCACGAGG CCATGGGCACCGGAGACCTGGACCTCCTATCAGCCTTCCGCCGCACGAGC CTGGCTGGAGGTGGTCGGCGGATCAGTGATAGCCATGAGGACACTGGGAT TCTGGACTTCAGCTCACTGCTGAAAAAGAGAGACAGTTTCCGGACCCCGA GGGACTCGAAGCTGGAGGCACCAGCAGAGGAGGACGTGTGGGAGATCCTA CGGCAGGCACCCCCATCTGAGTACGAGCGCATCGCCTTCCAGTACGGCGT CACTGACCTGCGCGGCATGCTAAAGAGGCTCAAGGGCATGAGGCGCGATG AGAAGAAGAGCACAGCCTTTCAGAAGAAGCTGGAGCCGGCCTACCAGGTG AGCAAAGGCCACAAGATCCGGCTGACCGTGGAACTGGCTGACCATGACGC TGAGGTCAAATGGCTCAAGAATGGCCAGGAGATCCAGATGAGCGGCAGCA AGTACATCTTTGAGTCCATCGGTGCCAAGCGTACCCTGACCATCAGCCAG TGCTCATTGGCGGACGACGCAGCCTACCAGTGCGTGGTGGGTGGCGAGAA GTGTAGCACGGAGCTCTTTGTGAAAGAGCCCCCTGTGTACCAGAGCATCG CAGACCTGATGGTGGGCGCAAAGGACGAGGCGGTGTTCAAATGTGAGGTC TCAGATGAGAATGTTCGGGGTGTGTGGCTGAAGAATGGGAAGGAGCTGGT GCCCGACAGCCGCATAAAGGTGTCCCACATCGGGCGGGTCCACAAACTGA CCATTGACGACGTCACACCTGCCGACGAGGCTGACTACAGCTTTGTGCCC GAGGGCTTCGCCTGCAACCTGTCAGCCAAGCTCCACTTCATGGAGGTCAA GATTGACTTCGTACCCAGGCAGGAACCTCCCAAGATCCACCTGGACTGCC CAGGCCGCATACCAGACACCATTGTGGTTGTAGCTGGAAATAAGCTACGT CTGGACGTCCCTATCTCTGGGGACCCCGCTCCCACTGTGATCTGGCAGAA GGCTATCACGCAGGGGAATAAGGCCCCAGCCAGGCCAGCCCCAGATGCCC CAGAGGACACAGGTGACAGCGATGAGTGGGTGTTTGACAAGAAGCTGCTG TGTGAGACCGAGGGCCGGGTCCGCGTGGAGACCACCAAGGACCGCAGCAT CTTCACGGTCGAGGGGGCAGAGAAGGAAGATGAGGGCGTCTACACGGTCA CAGTGAAGAACCCTGTGGGCGAGGACCAGGTCAACCTCACAGTCAAGGTC ATCGACGTGCCAGACGCACCTGCGGCCCCCAAGATCAGCAACGTGGGAGA GGACTCCTGCACAGTACAGTGGGAGCCGCCTGCCTACGATGGCGGGCAGC CCATCCTGGGCTACATCCTGGAGCGCAAGAAGAAGAAGAGCTACCGGTGG ATGCGGCTGAACTTCGACCTGATTCAGGAGCTGAGTCATGAAGCGCGGCG CATGATCGAGGGCGTGGTGTACGAGATGCGCGTCTACGCGGTCAACGCCA TCGGCATGTCCAGGCCCAGCCCTGCCTCCCAGCCCTTCATGCCTATCGGT CCCCCCAGCGAACCCACCCACCTGGCAGTAGAGGACGTCTCTGACACCAC GGTCTCCCTCAAGTGGCGGCCCCCAGAGCGCGTGGGAGCAGGAGGCCTGG ATGGCTACAGCGTGGAGTACTGCCCAGAGGGCTGCTCAGAGTGGGTGGCT GCCCTGCAGGGGCTGACAGAGCACACATCGATACTGGTGAAGGACCTGCC CACGGGGGCCCGGCTGCTTTTCCGAGTGCGGGCACACAATATGGCAGGGC CTGGAGCCCCTGTTACCACCACGGAGCCGGTGACAGTGCAGGAGATCCTG CAACGGCCACGGCTTCAGCTGCCCAGGCACCTGCGCCAGACCATTCAGAA GAAGGTCGGGGAGCCTGTGAACCTTCTCATCCCTTTCCAGGGCAAGCCCC GGCCTCAGGTGACCTGGACCAAAGAGGGGCAGCCCCTGGCAGGCGAGGAG GTGAGCATCCGCAACAGCCCCACAGACACCATCCTGTTCATCCGGGCCGC TCGCCGCGTGCATTCAGGCACTTACCAGGTGACGGTGCGCATTGAGAACA TGGAGGACAAGGCCACGCTGGTGCTGCAGGTTGTTGACAAGCCAAGTCCT CCCCAGGATCTCCGGGTGACTGACGCCTGGGGTCTTAATGTGGCTCTGGA GTGGAAGCCACCCCAGGATGTCGGCAACACGGAACTCTGGGGGTACACAG TGCAGAAAGCCGACAAGAAGACCATGGAGTGGTTCACCGTCTTGGAGCAT TACCGCCGCACCCACTGCGTGGTGCCAGAGCTCATCATTGGCAATGGCTA CTACTTCCGCGTCTTCAGCCAGAATATGGTTGGCTTTAGTGACAGAGCGG CCACCACCAAGGAGCCCGTCTTTATCCCCAGACCAGGCATCACCTATGAG CCACCCAACTATAAGGCCCTGGACTTCTCCGAGGCCCCAAGCTTCACCCA GCCCCTGGTGAACCGCTCGGTCATCGCGGGCTACACTGCTATGCTCTGCT GTGCTGTCCGGGGTAGCCCCAAGCCCAAGATTTCCTGGTTCAAGAATGGC CTGGACCTGGGAGAAGACGCCCGCTTCCGCATGTTCAGCAAGCAGGGAGT GTTGACTCTGGAGATTAGAAAGCCCTGCCCCTTTGACGGGGGCATCTATG TCTGCAGGGCCACCAACTTACAGGGCGAGGCACGGTGTGAGTGCCGCCTG GAGGTGCGAGTGCCTCAGTAA MYBPC3-ATGCCTGAGCCGGGGAAGAAGCCAG 88 delC4 TCTCAGCTTTTAGCAAGAAGCCACGGTCAGTGGAAGTGGCCGCAGGCAGC CCTGCCGTGTTCGAGGCCGAGACAGAGCGGGCAGGAGTGAAGGTGCGCTG GCAGCGCGGAGGCAGTGACATCAGCGCCAGCAAGAAGTACGGCCTGGCCA CAGAGGGCACACGGCATACGCTGACAGTGCGGGAAGTGGGCCCTGCCGAC CAGGGATCTTACGCAGTCATTGCTGGCTCCTCCAAGGTCAAGTTCGACCT CAAGGTCATAGAGGCAGAGAAGGCAGAGCCCATCCTGGCCCCTGCCCCTG CCCCTGCTGAGGCCACTGGAGCCCCTGGAGAAGCCCCGGCCCCAGCCGCT GAGCTGGGAGAAAGTGCCCCAAGTCCCAAAGGGTCAAGCTCAGCAGCTCT CAATGGTCCTACCCCTGGAGCCCCCGATGACCCCATTGGCCTCTTCGTGA TGCGGCCACAGGATGGCGAGGTGACCGTGGGTGGCAGCATCACCTTCTCA GCCCGCGTGGCCGGCGCCAGCCTCCTGAAGCCGCCTGTGGTCAAGTGGTT CAAGGGCAAATGGGTGGACCTGAGCAGCAAGGTGGGCCAGCACCTGCAGC TGCACGACAGCTACGACCGCGCCAGCAAGGTCTATCTGTTCGAGCTGCAC ATCACCGATGCCCAGCCTGCCTTCACTGGCAGCTACCGCTGTGAGGTGTC CACCAAGGACAAATTTGACTGCTCCAACTTCAATCTCACTGTCCACGAGG CCATGGGCACCGGAGACCTGGACCTCCTATCAGCCTTCCGCCGCACGAGC CTGGCTGGAGGTGGTCGGCGGATCAGTGATAGCCATGAGGACACTGGGAT TCTGGACTTCAGCTCACTGCTGAAAAAGAGAGACAGTTTCCGGACCCCGA GGGACTCGAAGCTGGAGGCACCAGCAGAGGAGGACGTGTGGGAGATCCTA CGGCAGGCACCCCCATCTGAGTACGAGCGCATCGCCTTCCAGTACGGCGT CACTGACCTGCGCGGCATGCTAAAGAGGCTCAAGGGCATGAGGCGCGATG AGAAGAAGAGCACAGCCTTTCAGAAGAAGCTGGAGCCGGCCTACCAGGTG AGCAAAGGCCACAAGATCCGGCTGACCGTGGAACTGGCTGACCATGACGC TGAGGTCAAATGGCTCAAGAATGGCCAGGAGATCCAGATGAGCGGCAGCA AGTACATCTTTGAGTCCATCGGTGCCAAGCGTACCCTGACCATCAGCCAG TGCTCATTGGCGGACGACGCAGCCTACCAGTGCGTGGTGGGTGGCGAGAA GTGTAGCACGGACCTCTTTGTGAAAGAGCCCCCTGTGCTCATCACGCGCC CCTTGGAGGACCAGCTGGTGATGGTGGGGCAGCGGGTGGAGTTTGAGTGT GAAGTATCGGAGGAGGGGGCGCAAGTCAAATGGCTGAAGGACGGGGTGGA GCTGACCCGGGAGGAGACCTTCAAATACCGGTTCAAGAAGGACGGGCAGA GACACCACCTGATCATCAACGAGGCCATGCTGGAGGACGCGGGGCACTAT GCACTGTGCACTAGCGGGGGCCAGGCGCTGGCTGAGCTCATTGTGCAGGA AAAGAAGCTGGAGCCTCCCAAGATCCACCTGGACTGCCCAGGCCGCATAC CAGACACCATTGTGGTTGTAGCTGGAAATAAGCTACGTCTGGACGTCCCT ATCTCTGGGGACCCCGCTCCCACTGTGATCTGGCAGAAGGCTATCACGCA GGGGAATAAGGCCCCAGCCAGGCCAGCCCCAGATGCCCCAGAGGACACAG GTGACAGCGATGAGTGGGTGTTTGACAAGAAGCTGCTGTGTGAGACCGAG GGCCGGGTCCGCGTGGAGACCACCAAGGACCGCAGCATCTTCACGGTCGA GGGGGCAGAGAAGGAAGATGAGGGCGTCTACACGGTCACAGTGAAGAACC CTGTGGGCGAGGACCAGGTCAACCTCACAGTCAAGGTCATCGACGTGCCA GACGCACCTGCGGCCCCCAAGATCAGCAACGTGGGAGAGGACTCCTGCAC AGTAGAGTGGGAGCCGCCTGCCTACGATGGCGGGCAGCCCATCCTGGGCT ACATCCTGGAGCGCAAGAAGAAGAAGAGCTACCGGTGGATGCGGCTGAAC TTCGACCTGATTCAGGAGCTGAGTCATGAAGCGCGGCGCATGATCGAGGG CGTGGTGTACGAGATGCGCGTCTACGCGGTCAACGCCATCGGCATGTCCA GGCCCAGCCCTGCCTCCCAGCCCTTCATGCCTATCGGTCCCCCCAGCGAA CCCACCCACCTGGCAGTAGAGGACGTCTCTGACACCACGGTCTCCCTCAA GTGGCGGCCCCCAGAGCGCGTGGGAGCAGGAGGCCTGGATGGCTACAGCG TGGAGTACTGCCCAGAGGGCTGCTCAGAGTGGGTGGCTGCCCTGCAGGGG CTGACAGAGCACACATCGATACTGGTGAAGGACCTGCCCACGGGGGCCCG GCTGCTTTTCCGAGTGCGGGCACACAATATGGCAGGGCCTGGAGCCCCTG TTACCACCACGGAGCCGGTGACAGTGCAGGAGATCCTGCAACGGCCACGG CTTCAGCTGCCCAGGCACCTGCGCCAGACCATTCAGAAGAAGGTCGGGGA GCCTGTGAACCTTCTCATCCCTTTCCAGGGCAAGCCCCGGCCTCAGGTGA CCTGGACCAAAGAGGGGCAGCCCCTGGCAGGCGAGGAGGTGAGCATCCGC AACAGCCCCACAGACACCATCCTGTTCATCCGGGCCGCTCGCCGCGTGCA TTCAGGCACTTACCAGGTGACGGTGCGCATTGAGAACATGGAGGACAAGG CCACGCTGGTGCTGCAGGTTGTTGACAAGCCAAGTCCTCCCCAGGATCTC CGGGTGACTGACGCCTGGGGTCTTAATGTGGCTCTGGAGTGGAAGCCACC CCAGGATGTCGGCAACACGGAACTCTGGGGGTACACAGTGCAGAAAGCCG ACAAGAAGACCATGGAGTGGTTCACCGTCTTGGAGCATTACCGCCGCACC CACTGCGTGGTGCCAGAGCTCATCATTGGCAATGGCTACTACTTCCGCGT CTTCAGCCAGAATATGGTTGGCTTTAGTGACAGAGCGGCCACCACCAAGG AGCCCGTCTTTATCCCCAGACCAGGCATCACCTATGAGCCACCCAACTAT AAGGCCCTGGACTTCTCCGAGGCCCCAAGCTTCACCCAGCCCCTGGTGAA CCGCTCGGTCATCGCGGGCTACACTGCTATGCTCTGCTGTGCTGTCCGGG GTAGCCCCAAGCCCAAGATTTCCTGGTTCAAGAATGGCCTGGACCTGGGA GAAGACGCCCGCTTCCGCATGTTCAGCAAGCAGGGAGTGTTGACTCTGGA GATTAGAAAGCCCTGCCCCTTTGACGGGGGCATCTATGTCTGCAGGGCCA CCAACTTACAGGGCGAGGCACGGTGTGAGTGCCGCCTGGAGGTGCGAGTG CCTCAGTAA MYBPC3- ATGCCTGAGCCGGGGAAGAAGCCAG 89delC4b TCTCAGCTTTTAGCAAGAAGCCACG GTCAGTGGAAGTGGCCGCAGGCAGCCCTGCCGTGTTCGAGGCCGAGACAG AGCGGGCAGGAGTGAAGGTGCGCTGGCAGCGCGGAGGCAGTGACATCAGC GCCAGCAACAAGTACGGCCTGGCCACAGAGGGCACACGGCATACGCTGAC AGTGCGGGAAGTGGGCCCTGCCGACCAGGGATCTTACGCAGTCATTGCTG GCTCCTCCAAGGTCAAGTTCGACCTCAAGGTGATAGAGGCAGAGAAGGCA GAGCCCATGCTGGCCCCTGCCCCTGCCCCTGCTGAGGCCACTGGAGCCCC TGGAGAAGCCCCGGCCCCAGCCGCTGAGCTGGGAGAAAGTGCCCCAAGTC CCAAAGGGTCAAGCTCAGCAGCTCTCAATGGTCCTACCCCTGGAGCCCCC GATGACCCCATTGGCCTCTTCGTGATGCGGCCACAGGATGGCGAGGTGAC CGTGGGTGGCAGCATCACCTTCTCAGCCCGCGTGGCCGGCGCCAGCCTCC TGAAGCCGCCTGTGGTCAAGTGGTTCAAGGGCAAATGGGTGGACCTGAGC AGCAAGGTGGGCCAGCACCTGCAGCTGCACGACAGCTACGACCGCGCCAG CAAGGTCTATCTGTTCGAGCTGCACATCACCGATGCCCAGCCTGCCTTCA CTGGCAGCTACCGCTGTGAGGTGTCCACCAAGGACAAATTTGACTGCTCC AACTTCAATCTCACTGTCCACGAGGCCATGGGCACCGGAGACCTGGACCT CCTATCAGCCTTCCGCCGCACGAGCCTGGCTGGAGGTGGTCGGCGGATCA GTGATAGCCATGAGGACACTGGGATTCTGGACTTCAGCTCACTGCTGAAA AAGAGAGACAGTTTCCGGACCCCGAGGGACTCGAAGCTGGAGGCACCAGC AGAGGAGGACGTGTGGGAGATCCTACGGCAGGCACCCCCATCTGAGTACG AGCGCATCGCCTTCCAGTACGGCGTCACTGACCTGCGCGGCATGCTAAAG AGGCTCAAGGGCATGAGGCGCGATGAGAAGAAGAGCACAGCCTTTCAGAA GAAGCTGGAGCCGGCCTACCAGGTGAGCAAAGGCCACAAGATCCGGCTGA CCGTGGAACTGGCTGACCATGACGCTGAGGTCAAATGGCTCAAGAATGGC CAGGAGATCCAGATGAGCGGCAGCAAGTACATCTTTGAGTCCATCGGTGC CAAGCGTACCCTGACCATCAGCCAGTGCTCATTGGCGGACGACGCAGCCT ACCAGTGCGTGGTGGGTGGCGAGAAGTGTAGCACGGAGCTCTTTGTGAAA GAGCCCCCTGTGCTCATCACGCGCCCCTTGGAGGACCAGCTGGTGATGGT GGGGCAGCGGGTGGAGTTTGAGTGTGAAGTATCGGAGGAGGGGGCGCAAG TCAAATGGCTGAAGGACGGGGTGGAGCTGACCCGGGAGGAGACCTTCAAA TACCGGTTCAAGAAGGACGGGCAGAGACACCACCTGATCATCAACGAGGC CATGCTGGAGGACGCGGGGCACTATGCACTGTGCACTAGCGGGGGCCAGG CGCTGGCTGAGCTCATTGTGCAGGAAAAGAAGCTGGAGCCCAGGCAGGAA CCTCCCAAGATCCACCTGGACTGCCCAGGCCGCATACCAGACACCATTGT GGTTGTAGCTGGAAATAAGCTACGTCTGGACGTCCCTATCTCTGGGGACC CCGCTCCCACTGTGATCTGGCAGAAGGCTATCACGCAGGGGAATAAGGCC CCAGCCAGGCCAGCCCCAGATGCCCCAGAGGACACAGGTGACAGCGATGA GTGGGTGTTTGACAAGAAGCTGCTGTGTGAGACCGAGGGCCGGGTCCGCG TGGAGACCACCAAGGACCGCAGCATCTTCACGGTCGAGGGGGCAGAGAAG GAAGATGAGGGCGTCTACACGGTCACAGTGAAGAACCCTGTGGGCGAGGA CCAGGTCAACCTCACAGTCAAGGTCATCGACGTGCCAGACGCACCTGCGG CCCCCAAGATCAGCAACGTGGGAGAGGACTCCTGCACAGTACAGTGGGAG CCGCCTGCCTACGATGGCGGGCAGCCCATCCTGGGCTACATCCTGGAGCG CAAGAAGAAGAAGAGCTACCGGTGGATGCGGCTGAACTTCGACCTGATTC AGGAGCTGAGTCATGAAGCGCGGCGCATGATCGAGGGCGTGGTGTACGAG ATGCGCGTCTACGCGGTCAACGCCATCGGCATGTCCAGGCCCAGCCCTGC CTCCCAGCCCTTCATGCCTATCGGTCCCCCCAGCGAACCCACCCACCTGG CAGTAGAGGACGTCTCTGACACCACGGTCTCCCTCAAGTGGCGGCCCCCA GAGCGCGTGGGAGCAGGAGGCCTGGATGGCTACAGCGTGGAGTACTGCCC AGAGGGCTGCTCAGAGTGGGTGGCTGCCCTGCAGGGGCTGACAGAGCACA CATCGATACTGGTGAAGGACCTGCCCACGGGGGCCCGGCTGCTTTTCCGA GTGCGGGCACACAATATGGCAGGGCCTGGAGCCCCTGTTACCACCACGGA GCCGGTGACAGTGCAGGAGATCCTGCAACGGCCACGGCTTCAGCTGCCCA GGCACCTGCGCCAGACCATTCAGAAGAAGGTCGGGGAGCCTGTGAACCTT CTCATCCCTTTCCAGGGCAAGCCCCGGCCTCAGGTGACCTGGACCAAAGA GGGGCAGCCCCTGGCAGGCGAGGAGGTGAGCATCCGCAACAGCCCCACAG ACACCATCCTGTTCATCCGGGCCGCTCGCCGCGTGCATTCAGGCACTTAC CAGGTGACGGTGCGCATTGAGAACATGGAGGACAAGGCCACGCTGGTGCT GCAGGTTGTTGACAAGCCAAGTCCTCCCCAGGATCTCCGGGTGACTGACG CCTGGGGTCTTAATGTGGCTCTGGAGTGGAAGCCACCCCAGGATGTCGGC AACACGGAACTCTGGGGGTACACAGTGCAGAAAGCCGACAAGAAGACCAT GGAGTGGTTCACCGTCTTGGAGCATTACCGCCGCACCCACTGCGTGGTGC CAGAGCTCATCATTGGCAATGGCTACTACTTCCGCGTCTTCAGCCAGAAT ATGGTTGGCTTTAGTGACAGAGCGGCCACCACCAAGGAGCCCGTCTTTAT CCCCAGACCAGGCATCACCTATGAGCCACCCAACTATAAGGCCCTGGACT TCTCCGAGGCCCCAAGCTTCACCCAGCCCCTGGTGAACCGCTCGGTCATC GCGGGCTACACTGCTATGCTCTGCTGTGCTGTCCGGGGTAGCCCCAAGCC CAAGATTTCCTGGTTCAAGAATGGCCTGGACCTGGGAGAAGACGCCCGCT TCCGCATGTTCAGCAAGCAGGGAGTGTTGACTCTGGAGATTAGAAAGCCC TGCCCCTTTGACGGGGGCATCTATGTCTGCAGGGCCACCAACTTACAGGG CGAGGCACGGTGTGAGTGCCGCCTGGAGGTGCGAGTGCCTCAGTAA

TABLE 4B Illustrative MYBPC3 Protein Sequences SEQ ID NameProtein Sequence NO. MYBPC3 MPEPGKKPVSAFSKKPRSVEVAAGS 103PAVFEAETERAGVKVRWQRGGSDIS ASNKYGLATEGTRHTLTVREVGPADQGSYAVIAGSSKVKFDLKVIEAEKA EPMLAPAPAPAEATGAPGEAPAPAAELGESAPSPKGSSSAALNGPTPGAP DDPIGLFVMRPQDGEVTVGGSITFSARVAGASLLKPPVVKWFKGKWVDLS SKVGQRLQLHDSYDRASKVYLFELHITDAQPAFTGSYRCEVSTKDKFDCS NFNLTVHEAMGTGDLDLLSAFRRTSLAGGGRRISDSHEDTGILDFSSLLK KRDSFRTPRDSKLEAPAEEDVWEILRQAPPSEYERIAFQYGVTDLRGMLK RLKGMRRDEKKSTAFQKKLEPAYQVSKGHKIRLTVELADHDAEVKWLKNG QEIQMSGSKYIFESIGAKRTLTISQCSLADDAAYQCVVGGEKCSTELEVK EPPVLITRPLEDQLVMVGQRVEFECEVSEEGAQVKWLKDGVELTREETFK YRFKKDGQPHHLIINEAMLEDAGHYALCTSGGQAIAELIVQEKKLEVYQS IADLMVGAKDQAVFKCEVSDENVRGVWLKNGKELVPDSRIKVSHIGRVHK LTIDDVTPADEADYSFVPEGFACNLSAKLHFMEVKIDFVPRQEPPKIHLD CPGRIPDTIVVVAGNKLRLDVPISGDPAPTVIWQKAITQGMKAPARPAPD APEDTGDSDEWVFDKKLLCETEGRVRVETTKDRSIFTVEGAEKEDEGVYT VTVKNPVGEDQVNLTVKVIDVPDAPAAPKISNVGEDSCTVQWEPPAYDGG QPILGYILERKKKKSYRWMRLNFDLIQELSHEARRMIEGVVYEMRVYAVN AIQMSRPSPASQPFMPIGPPSEPTHLAVEDVSDTTVSLKWRPPERVGAGG LDGYSVEYCPEGGSEWVAALQGLTEHTSILVKDLPTGARLLFRVRAHNMA GPGAPVTTTEPVTVQEILQRPRLQLPRHLRQTIQKKVGEPVNLLIPFQGK PRPQVTWTKEGQPLAGEEVSIRNSFTDTILFIRAARRVHSGTYQVTVRIE NMEDKATLVLQVVDKPSPPQDLRVTDAWGLNVALEWKPPQDVGNTELWGY TVQKADKKTMEWFTVLEHYRRTHCVVPELIIGNGYYFRVFSQNMVGFSDR AATTKEPVFIPRPGITYEPPNYKALDFSEAPSFTQPLVNRSVIAGYTAML CCAVRGSPKPKISWFKNGLDLGEDARFRMFSKQGVLTLEIRKPCPFDGGI YVCRATNLQGEARCECRLEVRVPQ MYBPC3-MPEPGKKPVSAFSKKPRSVEVAAGS 104 delC3 PAVFEAETERAGVKVRWQRGGSDISASNKYGLATEGTRHTLTVREVGPAD QGSYAVIAGSSKVKFDLKVIEAEKAEPMLAPAPAPAEATGAPGEAPAPAA ELGESAPSPKGSSSAALNGPTPGAPDDPIGLFVMRPQDGEVTVGGSITFS ARVAGASLLKPPVVKWFKGKWVDLSSKVGQHLQLHDSYDRASKVYLFELH ITDAQPAFTGSYRCEVSTKDKFDCSNFNLTVHEAMGTGDLDLLSAFRRTS LAGGGRRISDSHEDTGILDFSSLLKKRDSFRTPRDSKLEAPAEEDVWEIL RQAPPSEYERIAFQYGVTDLRGMLKRLKGMRRDEKKSTAFQKKLEPAYQV SKGHKIRLTVELADHDAEVKWLKNGQEIQMSGSKYIFESIGAKRTLTISQ CSLADDAAYQCVVGGEKCSTELFVKEPPVYQSIADLMVGAKDQAVFKCEV SDENVRGVWLKNGKELVPDSRIKVSHIGRVHKLTIDDVTPADEADYSFVP EGFACNLSAKLHFMEVKIDFVPRQEPPKIKLDCPGRIPDTIVVVAGNKLR LDVPISGDPAPTVIWQKAITQGKKAPARPAPDAPEDTGDSDEWVFDKKLL CETEGRVRVETTKDRSIFTVEGAEKEDEGVYTVTVKNPVGEDQVNLTVKV IDVPDAPAAPKISNVGEDSCTVQWEPPAYDGGQPILGYILERKKKKSYRW MRLNFDLIQELSHEARRMIEGVVYEMRVYAVNAIGMSRPSPASQPFMPIG PPSEPTHLAVEDVSDTTVSLKWRPPERVGAGGLDGYSVEYCPEGCSEWVA ALQGLTEHTSILVKDLPTGARLLFRVRAKNMAGPGAPVTTTEPVTVQEIL QRPRLQLPRHLRQTIQKKVGEPVNLLIPFQGKPRPQVTWTKEGQPLAGEE VSIRNSPTDTILFIPAARRVHSGTYQVTVRIENMEDKATLVLQVVDKPSP PQDLRVTDAWGLNVALEWKPPQDVGNTELWGYTVQKADKKTMEWFTVLEH YRRTHCVVPELIIGNGYYFRVFSQNMVGFSDRAATTKEPVFIPRPGITYE PPNYKALDFSEAPSFTQPLVNRSVTAGYTAMLCCAVRGSPKPKISWFKNG LDLGEDARFRMFSKQGVLTLEIRKPCPFDGGIYVCRATNLQGEARCECRL EVRVP MYBPC3- MPEPGKKPVSAFSKKPRSVEVAAGS 105delC4 PAVFEAETERAGVKVRWQRGGSDIS ASNKYGLATEGTRHTLTVREVGPADQGSYAVIAGSSKVKFDLKVIEAEKA EPMLAPAPAPAEATGAPGEAPAPAAELGESAPSPKGSSSAALNGPTPGAP DDPIGLFVMRPQDGEVTVGGSITFSARVAGASLLKPPVVKWFKGKWVDLS SKVGQHLQLHDSYDRASKVYLFELHITDAQPAFTGSYRCEVSTKDKFDCS NFNLTVHEAMGTGDLDLLSAFRRTSLAGGGRRISDSHEDTGILDFSSLLK KRDSFRTPRDSKLEAPAEEDVWEILRQAPPSEYERIAFQYGVTDLRGMLK RLKGMRRDEKKSTAFQKKLEPAYQVSKGHKIRLTVELADHDAEVKWLKNG QEIQMSGSKYIFESIGAKRTLTISQCSLADDAAYQCVVGGEKCSTELFVK EPPVLITRPLEDQLVMVGQRVEFECEVSEEGAQVKWLKDGVELTREETFK YRFKKDGQRHHLIINEAMLEDAGHYALCTSGGQALAELIVQEKKLEPPKI HLDCPGRIPDTIVVVAGNKLRLDVPISGDPAPTVIWQKAITQGNKAPARP APDAPEDTGDSDEWVFDKKLLCETEGRVRVETTKDRSIFTVEGAEKEDEG VYTVTVKWPVGEDQVNLTVKVIDVPDAPAAPKISNVGEDSCTVQWEPPAY DGGQPILGYILERKKKKSYRWMRLNBDLIQELSHEARRMIEGVVYEMRVY AVNAIGMSRPSPASQPFMPIGPPSEPTHLAVEDVSDTTVSLKWRPPERVG AGGLDGYSVEYCPEGCSEWVAALQGLTEKTSILVKDLPTGARLLFRVRAK NMAGPGAPVTTTEPVTVQEILQRPRLQLPRHLRQTIQKKVGEPVNLLIPF QGKPRPQVTWTKEGQPLAGEEVSIRNSPTDTILFIRAARRVHSGTYQVTV RIENMEDKATLVLQVVDKPSPPQDLRVTDAWGLNVALEWKPPQDVGNTEL WGYTVQKADKKTMEWFTVLEHYRRTHCVVPELIIGNGYYFRVFSQNKVGF SDRAATTKEPVFIPRPGITYEPPNYKALDFSEAPSFTQPLVNRSVIAGYT AMLCCAVRGSPKPKISWFKNGLDLGEDARFRMFSKQGVLTLEIRKPCPFD GGIYVCRATNLQGEARCECRLEVRV PQ MYBPC3-MPEPGKKPVSAFSKKPRSVEVAAGS 106 delC4b PAVFEAETERAGVKVRWQRGGSDISASNKYGLATEGTRHTLTVREVGPAD QGSYAVIAGSSKVKFDLKVIEAEKAEPMLAPAPAPAEATGAPGEAPAPAA ELGESAPSPKGSSSAALNGPTPGAPDDPIGLFVMRPQDGEVTVGGSITFS ARVAGASLLKPPVVKWFKGKWVDLSSKVGQHLQLHDSYDRASKVYLFELH ITDAQPAFTGSYRCEVSTKDKFDCSNFNLTVHEAMGTGDLDLLSAFRRTS LAGGGRRISDSHEDTGILDFSSLLKKRDSFRTPRDSKLEAPAEEDVWEIL RQAPPSEYERIAFQYGVTDLRGMLKRLKGMRRDEKKSTAFQKKLEPAYQV SKGHKIRLTVELADHDAEVKWLKNGQEIQMSGSKYIFESIGAKRTLTISQ CSLADDAAYQCVVGGEKCSTELFVKEPPVLITRPLEDQLVMVGQRVEFEC EVSEEGAQVKWLKDGVELTREETFKYRFKKDGQRHHLIINEAMLEDAGHY ALCTSGGQALAELIVQEKKLEPRQEPPKIHLDCPGRIPDTIVVVAGNKLR LDVPISGDPAPTVIWQKAITQGNKAPARPAPDAPEDTGDSDEWVFDKKLL CETEGRVRVETTKDRSIFTVEGAEKEDEGVYTVTVKNPVGEDQVNLTVKV IDVPDAPAAPKISNVGEDSCTVQWEPPAYDGGQPILGYILERKKKKSYRW MRLNFDLIQELSHEARRMIEGVVYEMRVYAVNAIGMSRPSPASQPFMPIG PPSEPTHLAVEDVSDTTVSLKWRPPERVGAGGLDGYSVEYCPEGCSEWVA ALQGLTEHTSILVKDLPTGARLLFRVRAHNMAGPGAPVTTTEPVTVQEIL QRPRLQLPRHLRQTIQKKVGEPVNLLIPFQGKPRPQVTWTKEGQPLAGEE VSIRNSPTDTILFIRAARRVHSGTYQVTVRIENMEDKATLVLQVVDKPSP PQDLRVTDAWGLNVALEWKPPQDVGNTELWGYTVQKADKKTMEWFTVLEH YRRTHCVVPELIIGNGYYFRVFSQNMVGFSDRAATTKEPVFIPRPGITYE PPNYKALDFSEAPSFTQPLVNRSVIAGYTAMLCCAVRGSPKPKISWFKNG LDLGEDARFRMFSKQGVLTLEIRKPCPFDGGIYVCRATNLQGEARCECRL EVRVP

In some embodiments, the disclosure provides a vector comprising apolynucleotide sequence that encodes the potassium voltage gated channelsubfamily H member 2 (KCNH2) protein, operatively linked to a modifiedcardiac TNNT2 promoter. Similarly stated, in some embodiments, thepolynucleotide sequence operatively linked to the cardiac-specificpromoter (e.g., a modified cardiac TNNT2 promoter) is KCNH2, or amutant, variant, or fragment thereof (e.g., SEQ ID NO. 107). In humans,the KCNH2 gene encodes the KCNH2 protein (also known as hERG1, e.g., SEQID NO: 108), which forms a potassium channel with other KCNH2 proteinsto transport potassium out of cells. KCHN2 proteins are abundantlyexpressed in cardiac muscle, which function to recharge the cardiactissue after each heartbeat to maintain regular rhythm. In someembodiments, the polynucleotide encoding KCNH2 shares at least 90%, 95%,99%, or 100% identity to SEQ ID NO: 107. In some embodiments, the KCNH2protein shares at least 90%, 95%, 99%, or 100% identity to SEQ ID NO:108.

In some embodiments, the disclosure provides a vector comprising apolynucleotide sequence that encodes the transient receptor potentialcation channel subfamily M membrane 4 (TRPM4) protein, operativelylinked to a modified cardiac TNNT2 promoter. Similarly stated, in someembodiments, the polynucleotide sequence operatively linked to thecardiac-specific promoter (e.g., a modified cardiac TNNT2 promoter) isTRPM4, or a mutant, variant, or fragment thereof (e.g., SEQ ID NO: 109).In humans, the TRPM4 gene encodes the TRPM4 protein (e.g., SEQ ID NO:110), which functions as a channel to control the flow of cations intoand out of cells. The TRPM4 channel is abundantly expressed in cardiaccells and plays a key role in generating and transmitting electricalsignals. In some embodiments, the polynucleotide encoding TRPM4 sharesat least 90%, 95%, 99%, or 100% identity to SEQ ID NO: 109. In someembodiments, the TRPM4 protein shares at least 90%, 95%, 99%, or 100%identity to SEQ ID NO: 110.

In some embodiments, the disclosure provides a vector comprising apolynucleotide sequence that encodes the desmoglein 2 (DSG2) protein,operatively linked to a modified cardiac TNNT2 promoter. Similarlystated, in some embodiments, the polynucleotide sequence operativelylinked to the cardiac-specific promoter (e.g., a modified cardiac TNNT2promoter) is DSG2, or a mutant, variant, or fragment thereof (e.g., SEQID NO: 111). In humans, the DSG2 gene encodes DSG2 protein (e.g., SEQ IDNO: 112), which is a transmembrane glycoprotein and component ofdesmosomes. Desmosomes are intercellular junctions that provide strongadhesion between cells giving mechanical strength to tissues. In someembodiments, the polynucleotide encoding DSG2 shares at least 90%, 95%,99%, or 100% identity to SEQ ID NO: 111. In some embodiments, the DSG2protein shares at least 90%, 95%, 99%, or 100% identity to SEQ ID NO:112.

In some embodiments, the disclosure provides a vector comprising apolynucleotide sequence that encodes the ATPase sarcoplasmic/endoplasmicreticulum calcium transporting 2 (ATP2A2) protein, operatively linked toa modified cardiac TNNT2 promoter. Similarly stated, in someembodiments, the polynucleotide sequence operatively linked to thecardiac-specific promoter (e.g., a modified TNNT2 promoter) is ATP2A2,or a mutant, variant, or fragment thereof (e.g., SEQ ID NO: 113). Inhumans, the ATP2A2 gene encodes sarco(endo)plasmic reticulumcalcium-ATPase 2 (SERCA2) protein (e.g., SEQ ID NO: 114), whichcatalyzes the hydrolysis of ATP coupled with the translocation ofcalcium from the cytosol into the sarcoplasmic reticulum lumen. Theregulation of calcium ions into and out of the sarcoplasmic reticulumassists with muscle contraction and relaxation. In some embodiments, thepolynucleotide encoding ATP2A2 shares at least 90%, 95%, 99%, or 100%identity to SEQ ID NO: 113. In some embodiments, the ATP2A2 proteinshares at least 90%, 95%, 99%, or 100% identity to SEQ ID NO: 114.

In some embodiments, the disclosure provides a vector comprising apolynucleotide sequence that encodes the calcium voltage-gated channelsubunit alpha 1C (CACNA1C) protein, operatively linked to a modifiedcardiac TNNT2 promoter. Similarly stated, in some embodiments, thepolynucleotide sequence operatively linked to the cardiac-specificpromoter (e.g., a modified TNNT2 promoter) is CACNA1C, or a mutant,variant, or fragment thereof (e.g., SEQ ID NO: 115). In humans, theCACNA1C gene encodes the alpha 1 subunit of a voltage-dependent calciumchannel protein (e.g., SEQ ID NO: 116), which functions to mediate theinflux of calcium ions into the cell upon membrane polarization. In someembodiments, the polynucleotide encoding CACNA1C shares at least 90%,95%, 99%, or 100% identity to SEQ ID NO: 115. In some embodiments, theCACNA1C protein shares at least 90%, 95%, 99%, or 100% identity to SEQID NO: 116.

In some embodiments, the disclosure provides a vector comprising apolynucleotide sequence that encodes the dystrophin (DMD) protein,operatively linked to a modified cardiac TNNT2 promoter. Similarlystated, in some embodiments, the polynucleotide sequence operativelylinked to the cardiac-specific promoter (e.g., a modified TNNT2promoter) is DMD, or a mutant, variant, or fragment thereof (e.g., SEQID NO: 117). In humans, the DMD gene encodes the DMD protein (e.g., SEQID NO: 118), which forms a component of the dystrophin-glycoproteincomplex (DGC). The DGC acts as an anchor, connecting the cytoskeletonwith the extracellular matrix, thereby strengthening muscle fibers andprotecting them from injury as muscles contract and relax. In someembodiments, the polynucleotide encoding DMD shares at least 90%, 95%,99%, or 100% identity to SEQ ID NO: 117. In some embodiments, the DMDprotein shares at least 90%, 95%, 99%, or 100% identity to SEQ ID NO:118.

In some embodiments, the disclosure provides a vector comprising apolynucleotide sequence that encodes the DM1 protein kinase (DMPK)protein, operatively linked to a modified cardiac TNNT2 promoter.Similarly stated, in some embodiments, the polynucleotide sequenceoperatively linked to the cardiac-specific promoter (e.g., a modifiedTNNT2 promoter) is DMPK, or a mutant, variant, or fragment thereof(e.g., SEQ ID NO: 119). In humans, the DMPK gene encodes myotonicdystrophy protein kinase protein (e.g., SEQ ID NO: 120), which plays animportant role in brain, muscle and heart development and homeostasis.Myotonic dystrophy protein kinase inhibits myosin phosphatase, whichplays a role in muscle tensing and relaxation. In some embodiments, thepolynucleotide encoding DMPK shares at least 90%, 95%, 99%, or 100%identity to SEQ ID NO: 119. In some embodiments, the DMPK protein sharesat least 90%, 95%, 99%, or 100% identity to SEQ ID NO: 120.

In some embodiments, the disclosure provides a vector comprising apolynucleotide sequence that encodes the ectopic P granules protein 5homolog (EPG5) protein, operatively linked to a modified cardiac TNNT2promoter. Similarly stated, in some embodiments, the polynucleotidesequence operatively linked to the cardiac-specific promoter (e.g., amodified TNNT2 promoter) is EPG5, or a mutant, variant, or fragmentthereof (e.g., SEQ ID NO: 121). In humans, the EPG5 gene encodes theEPG5 protein (e.g., SEQ ID NO: 122), which functions in autophagy topromote the interaction between autophagosomes and lysosomes. In someembodiments, the polynucleotide encoding EPG5 shares at least 90%, 95%,99%, or 100% identity to SEQ ID NO: 121. In some embodiments, the EPG5protein shares at least 90%, 95%, 99%, or 100% identity to SEQ ID NO:122.

In some embodiments, the disclosure provides a vector comprising apolynucleotide sequence that encodes the EvC ciliary complex subunit 1(EVC) protein, operatively linked to a modified cardiac TNNT2 promoter.Similarly stated, in some embodiments, the polynucleotide sequenceoperatively linked to the cardiac-specific promoter (e.g., a modifiedTNNT2 promoter) is EVC, or a mutant, variant, or fragment thereof (e.g.,SEQ ID NO: 123). In humans, the EVC gene encodes the EVC protein (e.g.,SEQ ID NO: 124), which is found primarily in cilia, and functions totransmit information between cells. The EVC protein also regulates SonicHedgehog, which plays a role in cell growth and differentiation. In someembodiments, the polynucleotide encoding EVC shares at least 90%, 95%,99%, or 100% identity to SEQ ID NO: 123. In some embodiments, the EVCprotein shares at least 90%, 95%, 99%, or 100% identity to SEQ ID NO:124.

In some embodiments, the disclosure provides a vector comprising apolynucleotide sequence that encodes the limbin protein, operativelylinked to a modified cardiac TNNT2 promoter. Similarly stated, in someembodiments, the polynucleotide sequence operatively linked to thecardiac-specific promoter (e.g., a modified TNNT2 promoter) is EVC2, ora mutant, variant, or fragment thereof (e.g., SEQ ID NO: 125). Inhumans, the EVC2 gene encodes the limbin protein (e.g., SEQ ID NO: 126).While the function of limbin is unknown, it is important for normalgrowth and development, particularly the development of bones and teeth.In some embodiments, the polynucleotide encoding limbin shares at least90%, 95%, 99%, or 100% identity to SEQ ID NO: 125. In some embodiments,the limbin protein shares at least 90%, 95%, 99%, or 100% identity toSEQ ID NO. 126.

In some embodiments, the disclosure provides a vector comprising apolynucleotide sequence that encodes the fibrillin-1 protein,operatively linked to a modified cardiac TNNT2 promoter. Similarlystated, in some embodiments, the polynucleotide sequence operativelylinked to the cardiac-specific promoter (e.g., a modified TNNT2promoter) is FBNJ, or a mutant, variant, or fragment thereof (e.g., SEQID NO: 127). In humans, the FBNJ gene encodes fibrillin-1 and asprosinproteins (e.g., SEQ ID NO: 128). Fibrillin-1 is a glycoprotein thatserves as a structural component of calcium-binding microfibrils, whichprovide force-bearing support in elastic and nonelastic connectivetissue throughout the body. Asprosin is a hormone normally secreted bywhite adipose tissue to regulate glucose homeostasis. In someembodiments, the polynucleotide encoding fibrillin-1 shares at least90%, 95%, 99%, or 100% identity to SEQ ID NO: 127. In some embodiments,the fibrillin-1 protein shares at least 90%, 95%, 99%, or 100% identityto SEQ ID NO: 128.

In some embodiments, the disclosure provides a vector comprising apolynucleotide sequence that encodes the neurofibromin (NF1) protein,operatively linked to a modified cardiac TNNT2 promoter. Similarlystated, in some embodiments, the polynucleotide sequence operativelylinked to the cardiac-specific promoter (e.g., a modified TNNT2promoter) is NF1, or a mutant, variant, or fragment thereof (e.g., SEQID NO: 129). In humans, the NF1 gene encodes the NF1 protein (e.g., SEQID NO: 130), which functions as a tumor suppressor and negativeregulator of the Ras signaling pathway that stimulates cell growth anddivision. In some embodiments, the polynucleotide encoding NF1 shares atleast 90%, 95%, 99%, or 100% identity to SEQ ID NO: 129. In someembodiments, the NF1 protein shares at least 90%, 95%, 99%, or 100%identity to SEQ ID NO: 130.

In some embodiments, the disclosure provides a vector comprising apolynucleotide sequence that encodes the sodium channel protein type 5subunit alpha (SCN5A) protein, operatively linked to a modified cardiacTNNT2 promoter. Similarly stated, in some embodiments, thepolynucleotide sequence operatively linked to the cardiac-specificpromoter (e.g., a modified TNNT2 promoter) is SCN5A, or a mutant,variant, or fragment thereof (e.g., SEQ ID NO: 131). In humans, theSCN5A gene encodes the SCN5A protein (e.g., SEQ ID NO: 132), which is atetrodotoxin-resistant voltage-gated sodium channel subunit. SCN5A isfound primarily in cardiac muscle and is responsible for the initialupstroke of the action potential in an electrocardiogram. In someembodiments, the polynucleotide encoding SCN5A shares at least 90%, 95%,99%, or 100% identity to SEQ ID NO: 131. In some embodiments, the SCN5Aprotein shares at least 90%, 95%, 99%, or 100% identity to SEQ ID NO:132.

In some embodiments, the disclosure provides a vector comprising apolynucleotide sequence that encodes the son of sevenless homolog 1(SOS1) protein, operatively linked to a modified cardiac TNNT2 promoter.Similarly stated, in some embodiments, the polynucleotide sequenceoperatively linked to the cardiac-specific promoter (e.g., a modifiedTNNT2 promoter) is SOS1, or a mutant, variant, or fragment thereof(e.g., SEQ ID NO: 133). In humans, the SOS1 gene encodes the SOS1protein (e.g., SEQ ID NO: 134), which functions as a component of atrimeric complex that participates in transduction signals from Ras toRac by promoting Rac-specific guanine nucleotide exchange factoractivity. In some embodiments, the polynucleotide encoding SOS1 sharesat least 90%, 95%, 99%, or 100% identity to SEQ ID NO: 133. In someembodiments, the SOS1 protein shares at least 90%, 95%, 99%, or 100%identity to SEQ ID NO: 134.

In some embodiments, the disclosure provides a vector comprising apolynucleotide sequence that encodes the natriuretic peptide receptor 1(NPR 1) protein, operatively linked to a modified cardiac TNNT2promoter. Similarly stated, in some embodiments, the polynucleotidesequence operatively linked to the cardiac-specific promoter (e.g., amodified TNNT2 promoter) is NPR1, or a mutant, variant, or fragmentthereof (e.g., SEQ ID NO: 135). The NPR1 gene in humans encodes the NPR1protein (also referred to as GC-A) (e.g., SEQ ID NO: 136), which is atransmembrane catalytic receptor with intracellular guanylyl cyclaseactivity. NPR1 serves as a receptor for both atrial and brainnatriuretic peptides, which are vasoactive hormones that play a key rolein cardiovascular homeostasis. In some embodiments, the polynucleotideencoding NPR1 shares at least 90%, 95%, 99%, or 100% identity to SEQ IDNO: 135. In some embodiments, the NPR1 protein shares at least 90%, 95%,99%, or 100% identity to SEQ ID NO: 136.

In some embodiments, the disclosure provides a vector comprising apolynucleotide sequence that encodes the receptor tyrosine-proteinkinase erbB-4 (ERBB4) protein, operatively linked to a modified cardiacTNNT2 promoter. Similarly stated, in some embodiments, thepolynucleotide sequence operatively linked to the cardiac-specificpromoter (e.g., a modified TNNT2 promoter) is ERBB4, or a mutant,variant, or fragment thereof (e.g., SEQ ID NO: 137). The ERBB4 gene inhumans encodes the ERBB4 protein in humans (e.g., SEQ ID NO: 138), whichis a transmembrane receptor in the epidermal growth factor family.Signaling through the ERBB4 receptor induces a variety of cellularresponses, including mitogenesis and differentiation. In someembodiments, the polynucleotide encoding ERBB4 shares at least 90%, 95%,99%, or 100% identity to SEQ ID NO: 137. In some embodiments, the ERBB4protein shares at least 90%, 95%, 99%, or 100% identity to SEQ ID NO:138.

In some embodiments, the disclosure provides a vector comprising apolynucleotide sequence that encodes vasoactive intestinal peptide(VIP), operatively linked to a modified TNNT2 promoter. Similarlystated, in some embodiments, the polynucleotide sequence operativelylinked to the cardiac-specific promoter (e.g., a modified TNNT2promoter) is VIP, or a mutant, variant, or fragment thereof (e.g., SEQID NO: 139). In humans, the H7P gene encodes the vasoactive intestinalpeptide (e.g., SEQ ID NO. 140), which functions as a neuromodulator andneurotransmitter. VIP is a potent vasodilator, regulates smooth muscleactivity, epithelial cell secretion and blood flow in thegastrointestinal tract. In some embodiments, the polynucleotide encodingVIP shares at least 90%, 95%, 99%, or 100% identity to SEQ ID NO: 139.In some embodiments, the VIP protein shares at least 90%, 95%, 99%, or100% identity to SEQ ID NO: 140.

In some embodiments, the disclosure provides a vector comprising apolynucleotide sequence that encodes the beta-myosin heavy chain(MyHC-β), operatively linked to a modified cardiac TNNT2 promoter.Similarly stated, in some embodiments, the polynucleotide sequenceoperatively linked to the cardiac-specific promoter (e.g., a modifiedTNNT2 promoter) is MYH7, or a mutant, variant, or fragment thereof(e.g., SEQ ID NO: 141). In humans, the MYH7 gene encodes the MyHC-βprotein (e.g., SEQ ID NO: 142), which is a hexameric, asymmetric motorforming the majority of the thick filaments in cardiac muscle. Theenzymatic activity of the ATPase in the myosin head hydrolyzes ATP,fueling the process of shortening sarcomeres in order to generateintraventricular pressure and power. In some embodiments, thepolynucleotide encoding MyHC-β shares at least 90%, 95%, 99%, or 100%identity to SEQ ID NO: 141. In some embodiments, the MyHC-β proteinshares at least 90%, 95%, 99%, or 100% identity to SEQ ID NO: 142.

III. Vectors

In some embodiments, the disclosure provides vectors for the treatmentor prevention of heart disease. In particular, the vectors describedherein comprise a cardiac-specific promoter operatively linked to apolynucleotide that encodes a therapeutic protein, wherein expression ofthe therapeutic protein treats a subject in need thereof (e.g., asubject having cardiomyopathy). For example, in some embodiments, thevector is an AAV-based vector comprising the cardiac TNNT2 promoteroperatively linked to a polynucleotide that encodes the MYBPC3 proteinfor the treatment or prevention of cardiomyopathy.

In some embodiments, the vector comprises, in addition to thecardiac-specific promoters (e.g., a modified cardiac troponin Tpromoter) and therapeutic gene products (e.g., MYBPC3 protein) describedherein, a marker gene that facilitates identification or selection ofcells that have been transfected, transduced or infected. Examples ofmarker genes include, but are not limited to, genes encoding fluorescentproteins, e.g., enhanced green fluorescent protein, Ds-Red (DsRed:Discosoma sp. red fluorescent protein (RFP); Bevis et al. (2002) Nat.Biotechnol. 20(11):83-87), yellow fluorescent protein, mCherry, andcyanofluorescent protein; and genes encoding proteins conferringresistance to a selection agent, e.g., a neomycin resistance gene, apuromycin resistance gene, a blasticidin resistance gene, and the like.

In some embodiments, the vector comprises a polynucleotide sequencehaving a size of at most about 4.0 kilobases, at most about 4.5kilobases, at most about 5 kilobases, at most about 5.1 kilobases, atmost about 5.2 kilobases, at most about 5.3 kilobases, at most about 5.4kilobases, or at most about 5.5 kilobases. In some embodiments, thevector comprises a polynucleotide sequence having a size of at mostabout 4.5 kilobases. In some embodiments, the vector comprises apolynucleotide sequence having a size of at most about 5 kilobases. Insome embodiments, the vector comprises a polynucleotide sequence havinga size of at most about 5.5 kilobases. In some embodiments, the vectorcomprises a polynucleotide sequence having a size of at most about 6kilobases.

Methods of introducing polynucleotides into a host cell are known in theart, and any known method can be used to introduce the polynucleotidesdescribed herein into a cell. Suitable methods include e.g., viral orbacteriophage infection, transfection, conjugation, protoplast fusion,lipofection, electroporation, calcium phosphate precipitation,polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediatedtransfection, liposome-mediated transfection, particle gun technology,calcium phosphate precipitation, direct micro injection,nanoparticle-mediated nucleic acid delivery, microfluidics deliverymethods, and the like.

A. Non-Viral Vectors

In some embodiments, the polynucleotides described herein are deliveredto a cell in a non-viral vector, such as a transposon, a nanoparticle(e.g., a lipid nanoparticle), a liposome, an exosome, an attenuatedbacterium, or a virus-like particle. In some embodiments, the non-viralvector is a mammalian virus-like particle. For example, mammalianvirus-like particle can be generated (e.g., by purification of the“empty” mammalian virus-like particle followed by ex vivo assembly ofthe mammalian virus-like particle with the desired cargo). The non-viralvector can also be engineered to incorporate targeting ligands to altertarget tissue specificity.

B. Viral Vectors

In some embodiments, the vector is a viral vector. In some embodiments,the viral vector is a retroviral vector, e.g., a lentiviral vector. Asused herein, the term “retrovirus” or “retroviral” refers an RNA virusthat reverse transcribes its genomic RNA into a linear double-strandedDNA copy and subsequently covalently integrates its genomic DNA into ahost genome. Retrovirus vectors are a common tool for gene delivery(Miller, Nature. 357: 455-460 (2000)). Once the virus is integrated intothe host genome, it is referred to as a “provirus.” The provirus servesas a template for RNA polymerase II and directs the expression of RNAmolecules encoded by the virus. In some embodiments, a retroviral vectoris altered so that it does not integrate into the host cell genome.

Illustrative retroviruses (family Retroviridae) include, but are notlimited to: (1) genus gammaretrovirus, such as, Moloney murine leukemiavirus (M-MuLV), Moloney murine sarcoma virus (MoMSV), murine mammarytumor virus (MuMTV), gibbon ape leukemia virus (GaLV), and felineleukemia virus (FLV), (2) genus spumavirus, such as, simian foamy virus,(3) genus lentivirus, such as, human immunodeficiency virus-1 and simianimmunodeficiency virus.

As used herein, the term “lentiviral” or “lentivirus” refers to a group(or genus) of complex retroviruses. Illustrative lentiviruses include,but are not limited to: HIV (human immunodeficiency virus; including HIVtype 1, and HIV type 2; visna-maedi virus (VMV) virus; the caprinearthritis-encephalitis virus (CAEV); equine infectious anemia virus(EIAV); feline immunodeficiency virus (FIV); bovine immune deficiencyvirus (BIV); and simian immunodeficiency virus (SIV).

In some embodiments, the viral vector is an adenoviral vector. Thegenetic organization of adenovirus includes an approximate 36 kb,linear, double-stranded DNA virus, which allows substitution of largepieces of adenoviral DNA with foreign sequences up to 7 kb (Grunhaus etal., Seminar in Virology 200(2):535-546, 1992)).

In some embodiments, the viral vector is an adeno-associated viral (AVV)vector, such as an AAV vector selected from the group consisting ofserotype 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 or chimeric AAV derivedthereof.

In some embodiments, the AAV expression vector is pseudotyped to enhancetargeting. A pseudotyping strategy can promote gene transfer and sustainexpression in a target cell type. For example, the AAV2 genome can bepackaged into the capsid of another AAV serotype such as AAV5, AAV7, orAAV8, producing pseudotyped vectors such as AAV2/5, AAV2/7, and AAV2/8respectively, as described in Balaji et al. J Surg Res. September;184(1): 691-698 (2013). In some embodiments, an AAV9 may be used totarget expression in myofibroblast-like lineages, as described in Piraset al. Gene Therapy 23:469-478 (2016). In some embodiments, AAV1, AAV6,or AAV9 is used, and in some embodiments, the AAV is engineered, asdescribed in Asokari et al. Hum Gene Ther. November; 24(11): 906-913(2013); Pozsgai et al. Mol Ther. April 5; 25(4): 855-869 (2017);Kotterman, M. A. and D. V. Schaffer Engineering Adeno-Associated Virusesfor Clinical Gene Therapy. Nature Reviews Genetics, 15:445-451 (2014);and US20160340393A1 to Schaffer et al. In some embodiments, the viralvector is AAV engineered to increase target cell infectivity asdescribed in US20180066285A1.

C Regulatory Elements

In some embodiments, the disclosure provides a vector comprising one ormore regulatory elements operatively linked to a polynucleotide encodinga therapeutic protein or nucleic acid. In some embodiments, theregulatory element is a cardiac-specific promoter (e.g., a modifiedTNNT2 promoter) that is operatively linked to a therapeutic protein ornucleic acid for the treatment of heart disease.

As used herein, the term “regulatory element” refers thosenon-translated regions of the vector (e.g., origin of replication,selection cassettes, promoters, enhancers, translation initiationsignals (Shine Dalgarno sequence or Kozak sequence) introns, apolyadenylation sequence, 5′ and 3′ untranslated regions) which interactwith host cellular proteins to carry out transcription and translation.Such elements may vary in their strength and specificity. Thetranscriptional regulatory element may be functional in either aeukaryotic cell (e.g., a mammalian cell) or a prokaryotic cell (e.g.,bacterial or archaeal cell). In some embodiments, a polynucleotidesequence encoding the therapeutic gene products (e.g., a therapeuticprotein or nucleic acid) described herein is operably linked to multiplecontrol elements that allow expression of the polynucleotide in bothprokaryotic and eukaryotic cells.

As used herein, the term “transcription start site” or “TSS” refers tothe first base pair transcribed by an RNA polymerase when the RNApolymerase initiates transcription. A TSS is different from the startcodon (canonically, ATG), which must be downstream of the TSS in thetranscribed region of the polynucleotide. The location of atranscription start site can be determined experimentally or byprediction using any of various prediction algorithms. Annotated TSSsare available from the Eukaryotic Promoter Database and the UCSC GenomeBrowser. Multiple TSSs for TNNT2 are identified in the UCSC GenomeBrowser.

As used herein, the TSS for TNNT2 is defined to be the sequenceidentified by the C at the 5′ end of the motif identified by dbTSS:CTCCATC.

The term “modified cardiac TNNT2 promoter” as used herein refers to apromoter that comprises a polynucleotide sequence of at least 200 basepairs that comprises one or more continuous or discontinuouspolynucleotide segments each sharing 70%, 80%, 90%, 95%, 96%, 97%, 98%,99%, or 100% identity to a corresponding segment of the TNNT2p-600segment provided in Table 1 as SEQ ID NO: 1. As it is a “promoter,” amodified cardiac TNNT2 promoter must be capable of promoting initiationof transcription by an RNA polymerase in a host or target cell at ornear a TSS within the promoter (i.e. at or near the TTS of TNNT2 asdefined herein) or, if the endogenous TSS of TNNT2 is not present in themodified cardiac TNNT2 promoter then at a heterologous TSS at most 100base pairs downstream (3′ on the sense strand) to the downstream (3′)end of the modified cardiac TNNT2 promoter. Similarly stated, a modifiedcardiac TNNT2 promoter may comprise only sequences upstream of the TSSof TNNT2 or more comprise the TSS of TNNT2.

The length of a promoter (e.g., a modified cardiac TNNT2 promoter), apromoter “having” so many base pairs, as used herein, is definedaccording to the number of base pairs in the polynucleotide sequence ofthe promoter from its 5′ end to its 3′ end, inclusive of the endpoints,and inclusive of any intervening sequences that do not align to areference promoter sequence (e.g., the endogenous cardiac TNNT2 promoterof a human or other organism). The 5′ end and the 3′ end of the promoterare defined as the last base pair in either direction to match acorresponding sequence in a reference promoter sequence when thesequence are aligned by the BLAST algorithm or the equivalent. Thus, thelength of a promoter in a vector can be determined by searching anucleotide database containing a genome of a reference organism usingthe polynucleotide sequence of the vector and identifying one or morealigned regions that encompass or are within about 1-5 kb of anendogenous gene, or by aligning the vector to a predetermined referencepromoter. If the promoter aligns to the reference genome or referencepromoter sequence as a continuous segment, then the length of promoteris the length alignment reported (the 3′ end position minus the 5′ endposition, +1 unless the TSS is included). If the promoter aligns inmultiple segments (e.g., 2, 3, 4, or 5 segments), then the length of thepromoter can be calculated by the 3′ end position of the 3′-most segmentof the reference genome or reference promoter sequence, minus the 5′ endposition of the 5′-most segment of reference genome or referencepromoter sequence, plus 1 unless the TSS is included (such that thecalculated length includes both end points). For example, the length ofa promoter that extends from a base pair 100 bp before the TSS (−100 bp)to 5 bp before the TSS (−5 bp) is −5−(−100)+1=100−5+1=96 bp. The TSS isnumbered +1 bp. Therefore, the length of a promoter that extends from abase pair 100 bp before the TSS (−100 bp) to 5 bp after the TSS (+5 bp)is +5−(−100)=100+5=105 bp.

The term “enhancer” refers to a segment of DNA which contains sequencescapable of providing enhanced transcription and in some instances canfunction independent of their orientation relative to another controlsequence. An enhancer can function cooperatively or additively withpromoters and/or other enhancer elements. An enhancer may overlap with apromoter or be upstream or downstream of the promoter. In someembodiments, the modified cardiac TNNT2 promoter comprises one or moreenhancers. In some embodiments, the modified cardiac TNNT2 promotercomprises no enhancer.

In addition to or instead of a modified cardiac TNNT2 promoter, someembodiments employ other eukaryotic promoters, including but not limitedto: cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV)thymidine kinase, a viral simian virus 40 (SV40) (e.g., early and lateSV40), a spleen focus forming virus (SFFV) promoter, long terminalrepeats (LTRs) from retrovirus (e.g., a Moloney murine leukemia virus(MoMLV) LTR promoter or a Rous sarcoma virus (RSV) LTR), a herpessimplex virus (HSV) (thymidine kinase) promoter, H5, P7.5, and P11promoters from vaccinia virus, an elongation factor 1-alpha (EF1α)promoter, early growth response 1 (EGR1) promoter, a ferritin H (FerH)promoter, a ferritin L (FerL) promoter, a Glyceraldehyde 3-phosphatedehydrogenase (GAPDH) promoter, a eukaryotic translation initiationfactor 4A1 (EIF4A1) promoter, a heat shock 70 kDa protein 5 (HSPA5)promoter, a heat shock protein 90 kDa beta, member 1 (HSP90B1) promoter,a heat shock protein 70 kDa (HSP70) promoter, a β-kinesin (β-KIN)promoter, the human ROSA 26 locus (Irions et al., Nature Biotechnology25, 1477-1482 (2007)), a Ubiquitin C (UBC) promoter, a phosphoglyceratekinase-1 (PGK) promoter, a cytomegalovirus enhancer/chicken β-actin(CAG) promoter, a β-actin promoter and a myeloproliferative sarcomavirus enhancer, negative control region deleted, dl587rev primer-bindingsite substituted (MND) promoter, and mouse metallothionein-1. The vectormay also contain a ribosome binding site for translation initiation anda transcription terminator. The vector may also include polynucleotidesequences for amplifying expression. The vector may also includepolynucleotide sequences encoding protein tags (e.g., 6×His tag,hemagglutinin tag, green fluorescent protein, etc.) that are fused tothe site-directed modifying polypeptide, thus resulting in a chimericpolypeptide.

In some embodiments, the promoters of the disclosure aretissue-specific. The term “tissue-specific promoter” means apolynucleotide sequence that serves as a promoter, i.e., regulatesexpression of a selected polynucleotide sequence operably linked to thepromoter, and which affects expression of the selected polynucleotidesequence in specific cells of a tissue, such as myocytes or myocardialcells. In some embodiments, the tissue-specific promoter is acardiac-specific promoter. In some embodiments, the cardiac-specificpromoter is TNNT2 or a modified TNNT2 promoter. A tissue-specificpromoter causes expression of an operatively linked polynucleotide, or agene product encoded by that polynucleotide, at 5×, 10×, 20×, 25× orgreater levels in the tissue of interest than in a reference tissue.

In some embodiments, the vectors described herein comprise atranscription termination signal. Elements directing the efficienttermination and polyadenylation of the heterologous nucleic acidtranscripts increases heterologous gene expression. Transcriptiontermination signals are generally found downstream of thepolyadenylation signal. In some embodiments, vectors comprise apolyadenylation sequence 3′ of a polynucleotide encoding a polypeptideto be expressed. The term “polyA site” or “polyA sequence” as usedherein denotes a DNA sequence which directs both the termination andpolyadenylation of the nascent RNA transcript by RNA polymerase II.Polyadenylation sequences can promote mRNA stability by addition of apolyA tail to the 3′ end of the coding sequence and thus, contribute toincreased translational efficiency. Cleavage and polyadenylation isdirected by a poly(A) sequence in the RNA. The core poly(A) sequence formammalian pre-mRNAs has two recognition elements flanking acleavage-polyadenylation site. Typically, an almost invariant AAUAAAhexamer lies 20-50 nucleotides upstream of a more variable element richin U or GU residues. Cleavage of the nascent transcript occurs betweenthese two elements and is coupled to the addition of up to 250adenosines to the 5′ cleavage product. In particular embodiments, thecore poly(A) sequence is an ideal polyA sequence (e.g., AATAAA, ATTAAA,AGTAAA). In particular embodiments, the poly(A) sequence is an SV40polyA sequence, a bovine growth hormone polyA sequence (BGHpA), a rabbitβ-globin polyA sequence (rβgpA), variants thereof, or another suitableheterologous or endogenous polyA sequence known in the art.

IV. Recombinant Adeno-Associated Virus (rAAV) Viral Genome, ExpressionCassette, and rAAV Virions

The disclosure provides an expression cassette comprising apolynucleotide encoding a transgene, e.g. a sequence encoding a MYBPC3polypeptide, or functional variant thereof. The transgene polynucleotidesequence in an expression cassette can be, for example, an open readingframe encoding a protein. The expression cassette may comprise,optionally, a promoter operatively linked to the transgene, optionallyan intron region, optionally a polyadenylation (poly(A)) signal,optionally a woodchuck hepatitis virus post-transcriptional element(WPRE), and optionally a transcription termination signal. Theexpression cassette may be flanked by one or more inverted terminalrepeats (ITRs). An expression cassette flanked by one or more ITRs isherein referred to as a “viral genome.” The ITRs in an expressioncassette serve as markers used for viral packaging of the expressioncassette (Clark et al. Hum Gene Ther. 6:1329-41 (1995)). Illustrativeand non-limiting embodiments of viral genomes of the disclosure areshown in FIG. 1A, FIG. 1C, and FIG. 2A. The polynucleotide encoding theexpression cassette provides the function of expressing the transgenewithin a host cell. The expression cassette can be integrated into thehost cell genome by, for example, infecting the host cell with an rAAVvirion comprising capsid protein and a viral genome comprising anexpression cassette.

The promoter sequence of the expression cassette, when present, controlsexpression of the polynucleotide encoding the transgene, e.g. a sequenceencoding MYBPC3 or functional variant thereof. Various promoters can beused. The promoter may be cell-type specific. Constitutive promoters areused in expression cassettes and can be, for example, thecytomegalovirus enhancer fused to the chicken β-actin promoter (CAG),simian virus 40 (SV40) promoter, and the herpes simplex virus thymidinekinase (HSV-TK) promoter (Damdindorj et al. PLoS One. 9:e106472 (2014)).Other cell-type specific promoters may also be used. Cardiac cellspecific promoters can be, for example, the MLC2v promoter (Phillips etal. Hypertension. 39:651-5 (2002)) and the cardiac Troponin-T (cTnT)promoter (Konkalmatt et al. Circ Cardiovasc Imaging. 6:478-486 (2013)).

In some aspects, the disclosure provides promoters have been optimizedfor cardiac cell-specific expression and length to accommodatetransgenes of specified size. In one embodiment, the promoter of an rAAVvector genome described herein is a polynucleotide having between 300 bpand 500 bp.

Exemplary expression cassette and viral genome sequences of thedisclosure can be found in Table 5. In some embodiments, the expressioncassette comprises a polynucleotide sequence that shares at least 90%,95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 95, SEQ ID NO:99, or SEQ ID NO: 101. In some embodiments, the viral genome comprises apolynucleotide sequence that shares at least 90%, 95%, 96%, 97%, 98%,99%, or 100% identity to SEQ ID NO: 98, SEQ ID NO: 100, or SEQ ID NO:102. In another embodiment, an expression cassette can be segmentedaccording to the polynucleotide regions flanking the transgene. Thepolynucleotide sequence spanning the 5′ end of the cassette to the 5′end of the transgene is herein referred to as the 5′ segment of theexpression cassette. The polynucleotide sequence spanning the 3′ end ofthe transgene to the 3′ end of the expression cassette is hereinreferred to as the 3′ segment of the expression cassette. In oneembodiment, the 5′ segment of the expression cassette comprises apolynucleotide sequence that shares at least 90%, 95%, 96%, 97%, 98%,99%, or 100% identity to SEQ ID NO: 93. In one embodiment, the 3′segment of the expression cassette comprises a polynucleotide sequencethat shares at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity toSEQ ID NO: 94.

The ability to express large transgenes delivered by rAAV vectors orrAAV virions is limited. rAAV vector genome sizes have a maximumsequence length of about 5 kb, thus providing a limit for the length ofall elements required in an expression cassette including regulatoryelements, e.g. promoter, and a transgene, e.g. MYBPC3. rAAV vectorgenomes exceeding 5 kb result in vector genome truncation during rAAVvirion packaging and reduce or ablate transgene expression (Wu et al.Mol Ther. 18:80-86 (2010)). In some embodiments, the present disclosureprovides rAAV vector genomes that are optimized for carrying largetransgenes. Elements of the vector genome have been reduced in length inorder to accommodate a larger transgene. In one embodiment, the 5′segment and 3′ segment of an expression cassette together comprise atmost 0.8 kbp or at most 0.9 kbp. In another embodiment, the 5′ ITR, the5′ segment, the 3′ segment, and 3′ ITR together comprise 1.2 kbp or atmost comprise 1.3 kbp. In one embodiment, the 5′ segment comprises atmost 500 bp or at most 480 bp. In one embodiment, the 3′ segmentcomprises at most 200 bp or at most 150 bp. In another embodiment, thevector genome comprises at most 4.7 kbp. 4.8 kbp, 4.9 kbp, or 5.0 kbp.In some embodiments, the polynucleotide encoding the gene productcomprises between 3 kb and 11 kb, between 3 kbp and 5 kbp, between 3.5kbp and 4.5 kbp, or between 3.7 kbp and 4 kbp. In some embodiments, thepolynucleotide encoding the gene product comprises 3.7 kbp to 3.9 kbp.In some embodiments, the polynucleotide encoding the gene productcomprises 3.8 kbp.

TABLE 5 Illustrative expression cassette and viral genomes SEQ ID NameDNA Sequence NO. viral Ctgcgcgctcgctcgctcactgag 98 genomegccgcccgggcaaagcccgggcgt (600 bp cgggcgacctttggtcgcccggcc promoter)tcagtgagcgagcgagcgcgcaga gagggagtggccaactccatcactaggggttccttgtagttaatgatt aacccgccatgctacttatctacgtagccatgctctaggaagatcgga attcgcccttaagtcatggagaagacccaccttgcagatgtcctcact ggggctggcagagccggcaacctgcccaaggctgctcagtccattagg agccagtagcctggaagatgtctttacccccagcatcagttcaagtgg agcagcacataactcttgccctctgccttccaagattctggtgctgag acttatggagtgtcttggaggttgccttctgccccccaaccctgctcc cagctggccctcccaggcctgggttgctggcctctgctttatcaggat tctcaagagggacagctggtttatgttgcatgactgttccctgcatat ctgctctggttttaaatagcttatctgagcagctggaggaccacatgg gcttatatggcgtggggtacatgttcctgtagccttgtccctggcacc tgccaaaatagcagccaacaccccccacccccaccgccatccccctgc cccacccgtcccctgtcgcacattcctccctccgcagggctggctcat caggccccagcccacatgcctgcttaaagccctctccatcctctgcct cacccagtccccgctgagactgagcagacgcctccagccaccaagctt aataaaagatctttattttcattagatctgtgtgttggttttttgtgt gctggggactcgagttaagggcgaattcccgataaggatcttcctaga gcatggctacgtagataagtagcatggcgggttaatcattaactacaa ggaacccctagtgatggagttggccactccctctctgcgcgctcgctc gctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgc ccgggcggcctcagtgagcgagcg agcgcgcag expressionTgtagttaatgattaacccgccat 99 cassette gctacttatctacgtagccatgct (600 bpctaggaagatcggaattcgccctt promoter) aagtcatggagaagacccaccttgcagatgtcctcactggggctggca gagccggcaacctgcccaaggctgctcagtccattaggagccagtagc ctggaagatgtctttacccccagcatcagttcaagtggagcagcacat aactcttgccctctgccttccaagattctggtgctgagacttatggag tgtcttggaggttgccttctgccccccaaccctgctcccagctggccc tcccaggcctgggttgctggcctctgctttatcaggattctcaagagg gacagctggtttatgttgcatgactgttccctgcatatctgctctggt tttaaatagcttatctgagcagctggaggaccacatgggcttatatgg cgtggggtacatgttcctgtagccttgtccctggcacctgccaaaata gcagccaacaccccccacccccaccgccatccccctgccccacccgtc ccctgtcgcacattcctccctccgcagggctggctcaccaggccccag cccacatgcctgcttaaagccctctccatcctctgcctcacccagtcc ccgctgagactgagcagacgcctccagccaccaagcttaataaaaaat ctttattttcattagatctgtgtgttggttttttgtgtgctggggact cgagttaagggcgaattcccgataaggatcttcctagagcatggctac gtagataagtagcatggcgggtta atcattaactacaa viralCtgcgcgctcgctcgctcactgag 100 genome gccgcccgggcaaagcccgggcgt (400 bpcgggcgacctttggtcgcccggcc promoter) tcagtgagcgagcgagcgcgcagagagggagtggccaactccatcact aggggttccttgtagttaatgattaacccgccatgctacttatctacg tagccatgctctaggaagatcggaattcgcccttaagttgccttctgc cccccaaccctgctcccagctggccctcccaggcctgggttgctggcc tctgctttatcaggattctcaagagggacagctggtttatgttgcatg actgttccctgcatatctgctctggttttaaatagcttatctgagcag ctggaggaccacatgggcttatatggcgtggggtacatgttcctgtag ccttgtccctggcacctgccaaaatagcagccaacaccccccaccccc accgccatccccctgccccacccgtcccctgtcgcacattcctccctc cgcagggctggctcaccaggccccagcccacatgcctgcttaaagccc tctccatcctctgcctcacccagtccccgctgagactgagcagacgcc tccagccaccaagcttaataaaagatctttattttcattagatctgtg tgttggttttttgtgtgctggggactcgagttaagggcgaattcccga taaggatcttcctagagcatggctacgtagataagtagcatggcgggt taatcattaactacaaggaacccctagtgatggagttggccactccct ctctgcgcgctcgctcgctcactgaggccgggcgaccaasggtcgccc gacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgca g expression Tgtagttaatgattaacccgccat 101cassette gctacttatctacgtagccatgct (400 bp ctaggaagatcggaattcgcccttpromoter) aagttgccttctgccccccaaccc tgctcccagctggccctcccaggcctgggttgctggcctctgctttat caggattctcaagagggacagctggtttatgttgcatgactgttccct gcatatctgctctggttttaaatagcttatctgagcagctggaggacc acatgggcttatatggcgtggggtacatgttcctgtagccttgtccct ggcacctgccaaaatagcagccaacaccccccacccccaccgccatcc ccctgccccacccgtcccctgtcgcacattcctccctccgcagggctg gctcaccaggccccagcccacatgcctgcttaaagccctctccatcct ctgcctcacccagtccccgctgagactgagcagacgcctccagccacc aagcttaataaaagatctttattttcattagatctgtgtgttggtttt ttgtgtgctggggactcgagttaagggcgaattcccgataaggatctt cctagagcatggctacgtagataagtagcatggcgggttaatcattaa ctaca viral ctgcgcgctcgctcgctcactgag 102genome + gccgcccgggcaaagcccgggcgt MYBPC3 cgggcgacctttggtcgcccggcctransgene tcagtgagcgagcgagcgcgcaga (400 bp gagggagtggccaactccatcactpromoter) aggggttccttgtagttaatgatt aacccgccatgctacttatctacgtagccatgctctaggaagatcgga attcgcccttaagttgccttctgccccccaaccctgctcccagctggc cctcccaggcctgggttgctggcctctgctttatcaggattctcaaga gggacagctggtttatgttgcatgactgttccctgcatatctgctctg gttttaaatagcttatctgagcagctggaggaccacacgggcttatat ggcgtggggtacatgttcctgtagccttgtccctggcacctgccaaaa tagcagccaacaccccccacccccaccgccatccccctgccccacccg tcccctgtcgcacattcctccctccgcagggctggctcaccaggcccc agcccacatgcctgcttaaagccctctccatcctctgcctcacccagt ccccgctgagactgagcagacgcctccagccaccatgcctgagccggg gaagaagccagtcccagcttttagcaagaagccacggtcagtggaagt ggccgcaggcagccctgccgtgttcgaggccgagacagagcgggcagg agtgaaggtgcgctggcagcgcggaggcagtgacatcagcgccagcaa caagtacggcctggccacagagggcacacggcatacgctgacagtgcg ggaagtgggccctgccgaccagggatcttacgcagtcattgctggctc ctccaaggtcaagttcgacctcaaggtcatagaggcagagaaggcaga gcccatgctggcccctgcccctgcccctgctgaggccactggagcccc tggagaagccccggccccagccgctgagctgggagaaagtgccccaag tcccaaagggtcaagctcagcagctctcaatggtcctacccctggagc ccccgatgaccccattggcctcttcgtgatgcggccacaggatggcga ggtgaccgtgggtggcagcatcaccttctcagcccgcgtggccggcgc cagcctcctgaagccgcctgtggtcaagtggttcaagggcaaatgggt ggacctgagcagcaaggtgggccagcacctgcagctgcacgacagcta cgaccgcgccagcaaggtctatctgttcgagctgcacatcaccgatgc ccagcctgccttcactggcagctaccgctgtgaggtgtccaccaagga caaatttgactgctccaacttcaatctcactgtccacgaggccatggg caccggagacctggacctcctatcagccttccgccgcacgagcctggc tggaggtggtcggcggatcagtgatagccatgaggacactgggattct ggacttcagctcactgctgaaaaagagagacagtttccggaccccgag ggactcgaagctggaggcaccagcagaggaggacgtgtgggagatcct acggcaggcacccccatctgagtacgagcgcatcgccttccagtacgg cgtcactgacctgcgcggcatgctaaagaggctcaagggcatgaggcg cgatgagaagaagagcacagcctttcagaagaagctggagccggccta ccaggtgagcaaaggccacaagatccggctgaccgtggaactggctga ccatgacgctgaggtcaaatggctcaagaatggccaggagatccagat gagcggcagcaagtacatctttgagtccatcggtgccaagcgtaccct gaccatcagccagtgctcattggcggacgacgcagcctaccagtgcgt ggtgggtggcgagaagtgtagcacggagctctttgtgaaagagccccc tgtgctcatcacgcgccccttggaggaccagctggtgatggtggggca gcgggtggagtttgagtgtgaagtatcggaggagggggcgcaagtcaa atggctgaaggacggggtggagctgacccgggaggagaccttcaaata ccggttcaagaaggacgggcagagacaccacctgatcatcaacgaggc catgctggaggacgcggggcactatgcactgtgcactagcgggggcca ggcgctggctgagctcattgtgcaggaaaagaagctggaggtgtacca gagcatcgcagacctgatggtgggcgcaaaggaccaggcggtgttcaa atgtgaggtctcagatgagaatgttcggggtgtgtggctgaagaatgg gaaggagctggtgcccgacagccgcataaaggtgtcccacatcgggcg ggtccacaaactgaccattgacgacgtcacacctgccgacgaggctga ctacagctttgtgcccgagggcttcgcctgcaacctgtcagccaagct ccacttcatggaggtcaagattgacttcgtacccaggcaggaacctcc caagatccacctggactgcccaggccgcataccagacaccattgtggt tgtagctggaaataagctacgtctggacgtccctatctctggggaccc cgctcccactgtgatctggcagaaggctatcacgcaggggaataaggc cccagccaggccagccccagatgccccagaggacacaggtgacagcga tgagtgggtgtttgacaagaagctgctgtgtgagaccgagggccgggt ccgcgtggagaccaccaaggaccgcagcatcttcacggtcgagggggc agagaaggaagatgagggcgtctacacggtcacagtgaagaaccctgt gggcgaggaccaggtcaacctcacagtcaaggtcatcgacgtgccaga cgcacctgcggcccccaagatcagcaacgtgggagaggactcctgcac agtacagtgggagccgcctgcctacgatggcgggcagcccatcctggg ctacatcctggagcgcaagaagaagaagagctaccggtggatgcggct gaacttcgacctgattcaggagctgagtcatgaagcgcggcgcatgat cgagggcgtggtgtacgagatgcgcgtctacgcggtcaacgccatcgg catgtccaggcccagccctgcctcccagcccttcatgcctatcggtcc ccccagcgaacccacccacctggcagtagaggacgtctctgacaccac ggtctccctcaagtggcggcccccagagcgcgtgggagcaggaggcct ggatggctacagcgtggagtactgcccagagggctgctcagagtgggt ggctgccctgcaggggctgacagagcacacatcgatactggtgaagga cctgcccacgggggcccggctgcttttccgagtgcgggcacacaatat ggcagggcctggagcccctgttacccccacggagccggtgacagtgca ggagatcctgcaacggccacggcttcagctgcccaggcacctgcgcca gaccattcagaagaaggtcggggagcctgtgaaccttctcatcccttt ccagggcaagccccggcctcaggtgacctggaccaaagaggggcagcc cctggcaggcgaggaggtgagcatccgcaacagccccacagacaccat cctgttcatccgggccgctcgccgcgtgcattcaggcacttaccaggt gacggtgcgcattgagaacatggaggacaaggccacgctggtgctgca ggttgttgacaagccaagtcctccccaggatctccgggtgactgacgc ctggggtcttaatgtggctctggagtggaagccaccccaggatgtcgg caacacggaactctgggggtacacagtgcagaaagccgacaagaagac catggagtggttcaccgtcttggagcattaccgccgcacccactgcgt ggtgccagagctcatcattggcaatggctactacttccgcgtcttcag ccagaatatggttggctttagtgacagagcggccaccaccaaggagcc cgtctttatccccagaccaggcatcacctatgagccacccaactataa ggccctggacttctccgaggccccaagcttcacccagcccctggtgaa ccgctcggtcatcgcgggctacactgctatgctctgctgtgctgtccg gggtagccccaagcccaagatttcctggttcaagaatggcctggacct gggagaagacgcccgcttccgcatgttcagcaagcagggagtgttgac tctggagattagaaagccctgcccctttgacgggggcatctatgtctg cagggccaccaacttacagggcgaggcacggtgtgagtgccgcctgga ggtgcgagtgcctcagtaaagcttaataaaagatctttattttcatta gatctgtgtgttggttttttgtgtgctggggactcgagttaagggcga attcccgataaggatcttcctagagcatggctacgtagataagtagca tggcgggttaatcattaactacaaggaacccctagtgatggagttggc cactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaa ggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcg agcgcgcag expression tgtagttaatgattaacccgccat95 cassette + gctacttatctacgtagccatgct MYBPC3 ctaggaagatcggaattcgccctttransgene aagttgccttctgccccccaaccc (400 bp tgctcccagctggccctcccaggcpromoter) ctgggttgctggcctctgctttat caggattctcaagagggacagctggtttatgttgcatgactgttccct gcatatctgctctggttttaaatagcttatctgagcagctggaggacc acatgggcttatatggcgtggggtacatgttcctgtagccttgtccct ggcacctgccaaaatagcagccaacaccccccacccccaccgccatcc ccctgccccacccgtcccctgtcgcacattcctccctccgcagggctg gctcaccaggccccagcccacatgcctgcttaaagccctctccatcct ctgcctcacccagtccccgctgagactgagcagacgcctccagccacc atgcctgagccggggaagaagccagtctcagcttttagcaagaagcca cggtcagtggaagtggccgcaggcagccctgccgtgttcgaggccgag acagagcgggcaggagtgaaggtgcgctggcagcgcggaggcagtgac atcagcgccagcaacaagtacggcctggccacagagggcacacggcat acgctgacagtgcgggaagtgggccctgccgaccagggatcttacgca gtcattgctggctcctccaaggtcaagttcgacctcaaggtcatagag gcagagaaggcagagcccatgctggcccctgcccctgcccctgctgag gccactggagcccctggagaagccccggccccagccgctgagctggga gaaagtgccccaagtcccaaagggtcaagctcagcagctctcaatggt cctacccctggagcccccgatgaccccattggcctcttcgtgatgcgg ccacaggatggcgaggtgaccgtgggtggcagcatcaccttctcagcc cgcgtggccggcgccagcctcctgaagccgcctgtggtcaagtggttc aagggcaaatgggtggacctgagcagcaaggtgggccagcacctgcag ctgcacgacagctacgaccgcgccagcaaggtctatctgttcgagctg cacatcaccgatgcccagcctgccttcactggcagctaccgctgtgag gtgtccaccaaggacaaatttgactgctccaacttcaatctcactgtc cacgaggccatgggcaccggagacctggacctcctatcagccttccgc cgcacgagcctggctggaggtggtcggcggatcagtgatagccatgag gacactgggattctggacttcagctcactgctgaaaaagagagacagt ttccggaccccgagggactcgaagctggaggcaccagcagaggaggac gtgtgggagatcctacggcaggcacccccatctgagtacgagcgcatc gccttccagtacggcgtcactgacctgcgcggcatgctaaagaggctc aagggcatgaggcgcgatgagaagaagagcacagcctttcagaagaag ctggagccggcctaccaggtgagcaaaggccacaagatccggctgacc gtggaactggctgaccatgacgctgaggtcaaatggctcaagaatggc caggagatccagatgagcggcagcaagtacatctttgagtccatcggt gccaagcgtaccctgaccatcagccagtgctcattggcggacgacgca gcctaccagtgcgtggtgggtggcgagaagtgtagcacggagctcttt gtgaaagagccccctgtgctcatcacgcgccccttggaggaccagctg gtgatggtggggcagcgggtggagtttgagtgtgaagtatcggaggag ggggcgcaagtcaaatggctgaaggacggggtggagctgacccgggag gagaccttcaaataccggttcaagaaggacgggcagagacaccacctg atcatcaacgaggccatgctggaggacgcggggcactatgcactgtgc actagcgggggccaggcgctggctgagctcattgtgcaggaaaagaag ctggaggtgtaccagagcatcgcagacctgatggtgggcgcaaaggac caggcggtgttcaaatgtgaggtctcagatgagaatgttcggggtgtg tggctgaagaatgggaaggagctggtgcccgacagccgcataaaggtg tcccacatcgggcgggtccacaaactgtccattgacgacgtcacacct gccgacgaggctgactacagctttgtgcccgagggcttcgcctgcaac ctgtcagccaagctccacttcatggaggtcaagattgacttcgtaccc aggcaggaacctcccaagatccacctggactgcccaggccgcatacca gacaccattgtggttgtagctggaaataagctacgtctggacgtccct atctctggggaccccgctcccactgtgatctggcagaaggctatcacg caggggaataaggccccagccaggccagccccagatgccccagaggac acaggtgacagcgatgagtgggtgtttgacaagaagctgctgtgtgag accgagggccgggtccgcgtggagaccaccaaggaccgcagcatcttc acggtcgagggggcagagaaggaagatgagggcgtctacacggtcaca gtgaagaaccctgtgggcgaggaccaggtcaacctcacagtcaaggtc atcgacgtgccagacgcacctgcggcccccaagatcagcaacgtggga gaggactcctgcacagtacagtgggagccgcctgcctacgatggcggg cagcccatcctgggctacatcctggagcgcaagaagaagaagagctac cggtggatgcggctgaacttcgacctgattcaggagctgagtcatgaa gcgcggcgcatgatcgagggcgtggtgtacgagatgcgcgtctacgcg gtcaacgccatcggcatgtccaggcccagccctgcctcccagcccttc atgcctatcggtccccccagcgaacccacccacctggcagtagaggac gtctctgacaccacggtctccctcaagtggcggcccccagagcgcgtg ggagcaggaggcctggatggctacagcgtggagtactgcccagagggc tgctcagagtgggtggctgccctgcaggggctgacagagcacacatcg atactggtgaaggacctgcccacgggggcccggctgcttttccgagtg cgggcacacaatatggcagggcctggagcccctgttaccaccacggag ccggtgacagtgcaggagatoctgcaacggccacggcttcagctgccc aggcacctgcgccagaccattcagaagaaggtcggggagcctgtgaac cttctcatccctttccagggcaagccccggcctcaggtgacctggacc aaagaggggcagcccctggcaggcgaggaggtgagcatccgcaacagc cccacagacaccatcctgttcatccgggccgctcgccgcgtgcattca ggcacttaccaggtgacggtgcgcattgagaacatggaggacaaggcc aogctggtgctgcaggttgttgacaagccaagtcctccccaggatctc cgggtgactgacgcctggggtcttaatgtggctctggagtggaagcca ccccaggatgtcggcaacacggaactctgggggtacacagtgcagaaa gccgacaagaagaccatggagtggttcaccgtcttggagcattaccgc cgcacccactgcgtggtgccagagctcatcattggcaatggctactac ttccgcgtcttcagccagaatatggttggctttagtgacagagcggcc accaccaaggagcccgtctttatccccagaccaggcatcacctatgag ccacccaactataaggccctggacttctccgaggccccaagcttcacc cagcccctggtgaaccgctcggtcatcgcgggctacactgctatgctc tgctgtgctgtccggggtagccccaagcccaagatttcctggttcaag aatggcctggacctgggagaagacgcccgcttccgcatgttcagcaag cagggagtgttgactctggagattagaaagccctgcccctttgacggg ggcatctatgtctgcagggccaccaacttacagggcgaggcacggtgt gagtgccgcctggaggtgcgagtgcctcagtaaagcttaataaaagat ctttattttcattagatctgtgtgttggttttttgtgtgctggggact cgagttaagggcgaattcccgataaggatcttcctagagcatggctac gtagataagtagcatggcgggtta atcattaactaca 5′tgtagttaatgattaacccgccat 93 segment- gctacttatctacgtagccatgct partialctaggaagatcggaattcgccctt viral aagttgccttctgccccccaaccc genometgctcccagctggccctcccaggc (400 bp ctgggttgctggcctctgctttat promoter)caggattctcaagagggacagctg gtttatgttgcatgactgttccctgcatatctgctctggttttaaata gcttatctgagcagctggaggaccacatgggcttatatggcgtggggt acatgttcctgtagccttgtccctggcacctgccaaaatagcagccaa caccccccacccccaccgccatccccctgccccacccgtcccctgtcg cacattcctccctccgcagggctggctcaccaggccccagcccacatg cctgcttaaagccctctccatcctctgcctcacccagtccccgctgag actgagcagacgcctccagccacc 3′agcttaataaaagatctttatttt 94 segment- cattagatctgtgtgttggttttt partialtgtgtgctggggactcgagttaag viral ggcgaattcccgataaggatcttc genomectagagcatggctacgtagataag tagcatggcgggttaatcattaac taca

In some aspects of the disclosure, an rAAV virion is used to deliver theexpression cassettes described herein to cardiac cells of a subject,e.g. to treat cardiomyopathy. Accordingly, the disclosure provides anrAAV virion, the rAAV virion comprising an AAV capsid and an expressioncassette comprising a polynucleotide encoding a transgene operativelylinked to a promoter.

The rAAV virions of the disclosure comprise a capsid protein. Capsidproteins are structural proteins that make up the assembled icosahedralpackaging of the rAAV virion that contains the expression cassette.Capsid proteins are classified by the serotype. Wild type capsidserotypes in rAAV virions can be, for example, AAV1, AAV2, AAV3, AAV4,AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12 (Naso et al.BioDrugs 31:317-334 (2017)). Engineered capsid types include chimericcapsids and mosaic capsids (Choi et al. Curr Gene Ther. 5: 299-310(2005)). Capsids are selected for rAAV virions based on their ability totransduce specific tissue or cell types (Liu et al. Curr Pharm Des.21:3248-56 (2015)).

Any capsid protein that can facilitate rAAV virion transduction intocardiac cells for delivery of a transgene, as described herein, can beused. Capsid proteins used in rAAV virions for transgene delivery tocardiac cells that result in high expression can be, for example, AAV4,AAV6, AAV7, AAV8, and AAV9 (Zincarelli et al. Mol. Ther. 16:P1073-1080(2008)). Artificial capsids, such as chimeric capsids generated throughcombinatorial libraries, can also be used for transgene delivery tocardiac cells that results in high expression (see U.S. 63/012,703, thecontents of which are herein incorporated by reference). Other capsidproteins with various features can also be used in the rAAV virions ofthe disclosure. AAV vectors and capsids are provided in U.S. Pat. Nos.10,011,640B2; 7,892,809B2, 8,632,764B2, 8,889,641B2, 9,475,845B2,10,889,833B2, 10,480,011B2, and 10,894,949B2, the contents of which areherein incorporated by reference; and Int'l Pat. Pub. Nos.WO2020198737A1, WO2019028306A2, WO2016054554A1, WO2018152333A1,WO2017106236A1, WO2008124724A1, WO2017212019A1, WO2020117898A1,WO2017192750A1, WO2020191300A1, and WO2017100671A1, the contents ofwhich are herein incorporated by reference.

In some embodiments, the rAAV virions of the disclosure comprise anengineered capsid protein. Engineered capsid proteins can be derivedfrom a parental, e.g. wild type, capsid and include, for example,variant polypeptide sequence with respect to a parental capsid sequenceat one or more sites. For example, variant sites of the parental capsidcan occur at the VR-IV site, VR-V site, VR-VII site and/or VR-VIII site(see, e.g. Boning and Srivastava. Mol Ther Methods Clin Dev. 12:248-265(2019)).

In some embodiments, the capsid protein is an AAV5/AAV9 chimeric capsidprotein. In some embodiments, the chimeric capsid protein comprises atleast 1, 2, 3, 4, 5 or more polypeptide segments that are derived fromAAV5 capsid protein (SEQ ID NO. 144). In some embodiments, the chimericcapsid protein comprises at least 1, 2, 3, 4, 5 or more polypeptidesegments that are derived from AAV9 capsid protein (SEQ ID NO: 143). Insome embodiments, at least one polypeptide segment is derived from theAAV5 capsid protein and at least one polypeptide segment is derived fromthe AAV9 capsid protein.

In some embodiments, the capsid protein is a combinatory capsidproteins. As used herein, “combinatory capsid protein” refers to aAAV5/AAV9 chimeric capsid protein, which further comprises amino acidvariations with respect to the chimeric parental sequence at one or moresites. In some embodiments, the one or more sites of the chimericparental sequence are selected from those equivalent to the VR-IV site,the VR-V site, the VR-VII site and the VR-VIII site of the AAV9 capsidprotein.

In some embodiments, the rAAV virions comprise an engineered capsidprotein selected from Table 6.

TABLE 6 Engineered Capsid Proteins Engineered Capsid SEQ ID NO: CR9-01145 CR9-07 146 CR9-08 147 CR9-09 148 CR9-10 149 CR9-11 150 CR9-13 151CR9-14 152 CR9-15 153 CR9-16 154 CR9-17 155 CR9-20 156 CR9-21 157 CR9-22158 ZC23 159 ZC24 160 ZC25 161 ZC26 162 ZC27 163 ZC28 164 ZC29 165 ZC30166 ZC31 167 ZC32 168 ZC33 169 ZC34 170 ZC35 171 ZC40 172 ZC41 173 ZC42174 ZC43 175 ZC44 176 ZC45 177 ZC46 178 ZC47 179 ZC48 180 ZC49 181 ZC50182 TN47-07 183 TN47-10 184 TN47-13 185 TN47-14 186 TN47-17 187 TN47-22188 TN40-07 189 TN40-10 190 TN40-13 191 TN40-14 192 TN40-17 193 TN40-22194 TN44-07 195 TN44-10 196 TN44-13 197 TN44-14 198 TN44-17 199 TN44-22200

In some embodiments, the rAAV is replication defective, in that the rAAVvirion cannot independently further replicate and package its genome.For example, when a cardiac cell is targeted with rAAV virions, thetransgene is expressed in the targeted cardiac cell, however, due to thefact that the targeted cardiac cell lacks AAV rep and cap genes andaccessory function genes, the rAAV is not able to replicate.

In some embodiments, rAAV virions of the present disclosureencapsulating the expression cassettes as described herein, can beproduced using helper-free production. rAAVs are replication-deficientviruses and normally require components from a live helper virus, suchas adenovirus, in a host cell for packaging of infectious rAAV virions.rAAV helper-free production systems allow the production of infectiousrAAV virions without the use of a live helper virus. In the helper-freesystem, a host packaging cell line is co-transfected with threeplasmids. A first plasmid may contain adenovirus gene products (e.g.E2A, E4, and VA RNA genes) needed for the packaging of rAAV virions. Asecond plasmid may contain required AAV genes (e.g., REP and CAP genes).A third plasmid contains the polynucleotide sequence encoding thetransgene of interest and a promoter flanked by ITRs. A host packagingcell line can be, for example, AAV-293 host cells. Suitable host cellscontain additional components required for packaging infectious rAAVvirions that are not supplied by the plasmids. In some embodiments, theCAP genes can encode, for example, AAV capsid proteins as describedherein.

IV. Methods of Treatment

The present disclosure also provides pharmaceutical compositionscomprising the rAAV vector genomes or rAAV virions disclosed herein andone or more pharmaceutically acceptable carriers, diluents orexcipients. In particular embodiments, the pharmaceutical compositioncomprises an rAAV vector genome or rAAV virion as described herein,comprising a polynucleotide sequence that encodes a therapeutic proteinor nucleic acid, operatively linked to a cardiac-specific promoter(e.g., a modified TNNT2 promoter). For example, in some embodiments, thepharmaceutical composition is an AAV9 vector comprising the modifiedcardiac TNNT2 promoter (SEQ ID NO: 3) operatively linked to the MYBPC3protein (SEQ ID NO: 86). Provided are pharmaceutical compositions, e.g.,for use in preventing or treating cardiomyopathy, which comprises atherapeutically effective amount of a vector that comprises apolynucleotide sequence encoding a therapeutic protein or nucleic acidthat can restore contractile function in the heart.

The disclosure provides methods for expressing a polynucleotide a cell.The method may comprise, for example, transducing a target cell with therAAV virions, rAAV vector genomes, or expression cassettes describedherein. A target cell can be, for example and without limitation, acardiac cell, a muscle cell, an induced pluripotent stem cell-derivedcardiomyocyte (iPSC-CM), a cardiomyocyte, or a MYBPC3^(−/−) iPSC-CM. Inone aspect, a method of expressing a MYBPC3 protein in a cell comprisestransducing a target cell or population of target cells with an rAAVvirion or rAAV vector genomes described herein. In one embodiment, thecell is a MYBPC3^(−/−) cell. In one embodiment, the cell comprises aninactivating mutation in one or both copies of the endogenous MYBPC3gene.

The compositions that are described herein can be employed in a methodof treating a subject with a cardiac disease or condition. “Treating” or“treatment of a condition or subject in need thereof” refers to (1)taking steps to obtain beneficial or desired results, including clinicalresults such as the reduction of symptoms; (2) preventing the disease,for example, causing the clinical symptoms of the disease not to developin a patient that may be predisposed to the disease, but does not yetexperience or display symptoms of the disease; (3) inhibiting thedisease, for example, arresting or reducing the development of thedisease or its clinical symptoms; (4) relieving the disease, forexample, causing regression of the disease or its clinical symptoms; or(5) delaying the disease. For purposes of this invention, beneficial ordesired clinical results include, but are not limited to, promotingcardiac sarcomere contraction.

Subjects in need of treatment using the compositions and methods of thepresent disclosure include, but are not limited to, individuals having acongenital heart defect, individuals suffering from a degenerativemuscle disease, individuals suffering from a condition that results inischemic heart tissue (e.g., individuals with coronary artery disease),and the like. In some examples, a method is useful to treat adegenerative muscle disease or condition (e.g., familial cardiomyopathy,dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictivecardiomyopathy, or coronary artery disease with resultant ischemiccardiomyopathy). In some examples, a subject method is useful to treatindividuals having a cardiac or cardiovascular disease or disorder, forexample, cardiovascular disease, aneurysm, angina, arrhythmia,atherosclerosis, cerebrovascular accident (stroke), cerebrovasculardisease, congenital heart disease, congestive heart failure,myocarditis, valve disease coronary, artery disease dilated, diastolicdysfunction, endocarditis, high blood pressure (hypertension),cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy,coronary artery disease with resultant ischemic cardiomyopathy, mitralvalve prolapse, myocardial infarction (heart attack), or venousthromboembolism. In some examples, the subject is suffering from or atrisk for cardiomyopathy.

In some embodiments, the compositions and methods disclosed herein canbe used for the prevention and/or treatment of cardiomyopathies in asubject. In some embodiments, the compositions and methods describedherein can be used to treat cardiomyopathies affiliated with mutationsin cardiac myosin binding protein C (MYBPC3), such as hypertrophiccardiomyopathy and familial hypertrophic cardiomyopathy. Thecardiomyopathy treated by the compositions and methods described hereincan also include cardiomyopathies associated with a pulmonary embolus, avenous thrombosis, a myocardial infarction, a transient ischemic attack,a peripheral vascular disorder, atherosclerosis, ischemic cardiacdisease and/or other myocardial injury or vascular disease. In certainembodiments, the cardiomyopathies treated by the compositions andmethods described herein can include cardiac diseases associated withmyocardial tissue hypercontractility, such as heart failure related toleft ventricular hypercontractility.

In some embodiments, the compositions and methods described herein caninduce detectable expression of a therapeutic protein or nucleic acid(e.g., MYBPC3 protein), or a mutant, variant, or fragment thereof, tomodulate contractile function of the myocardial tissue in a subject inneed thereof. In some embodiments, the amount, concentration, and volumeof the composition that modulates contractile function in myocardialtissue administered to a subject can be controlled and/or optimized tosubstantially improve the functional parameters of the heart whilemitigating adverse side effects.

The amount of the composition that modulates contractile functionadministered to myocardial tissue can also be an amount required toresult in the detectable expression of a therapeutic protein or nucleicacid (e.g., MYBPC3 protein) or a mutant, variant, or fragment thereof inthe heart; preserve and/or improve contractile function; delay theemergence of cardiomyopathy or reverse the pathological course of thedisease; increase myocyte viability; improve myofilament function;inhibit left ventricular hypertrophy; cardiac hypertrophy regression,normalize systolic and diastolic function in heart; and restore normalcross-bridge behavior at the myofilament level.

In some embodiments, the compositions and methods disclosed hereinresults in detectable expression of MYBPC3 protein, or a mutant,variant, or fragment thereof, in a cardiac cell of the subject beingtreated. In some embodiments, administration of an rAAV vector genome orrAAV virion described herein causes specific expression of MYBPC3protein in the heart of the subject. In some embodiments, administrationof rAAV vector genome or rAAV virion described herein causes low orundetectable expression of MYBPC3 in the skeletal tissue, brain, and/orliver of the subject, wherein optionally the subject suffers from or isat risk for cardiomyopathy.

In some embodiments, the compositions and methods disclosed hereinresults in detectable expression of KCNH2 protein, or a mutant, variant,or fragment thereof, in a cardiac cell of the subject being treated.

In some embodiments, the compositions and methods disclosed hereinresults in detectable expression of TRPM4 protein, or a mutant, variant,or fragment thereof, in a cardiac cell of the subject being treated.

In some embodiments, the compositions and methods disclosed hereinresults in detectable expression of DSG2 protein, or a mutant, variant,or fragment thereof, in a cardiac cell of the subject being treated.

In some embodiments, the compositions and methods disclosed hereinresults in detectable expression of ATP2A2 protein, or a mutant,variant, or fragment thereof, in a cardiac cell of the subject beingtreated.

In some embodiments, the compositions and methods disclosed hereinresults in detectable expression of CACNA1C, DMD, DMPK, EPG5, EVC, EVC2,FBN1, NF1, SCN5A, SOS1, NPR1, ERBB4, VIP, or MYH7, or a mutant, variant,or fragment thereof, in a cardiac cell of the subject being treated.

“Detectable expression” typically refers to expression at least 5%, 10%,15%, 20% or more compared to a control subject or tissue not treatedwith the vector. In some embodiments, detectable expression meansexpression at 1.5-fold, 2-fold, 2.5-fold, or 3-fold greater than ano-vector control. Expression can be assess by Western blot, asdescribed in the example that follows, or enzyme-linked immunosorbentassay (ELISA), or other methods known in the art. In some cases,expression is measured quantitatively using a standard curve. Standardcurves can be generated using purified protein, e.g. purified MYBPC3protein, by methods described in the examples or known in the art.Alternatively, expression of the therapeutic gene product can beassessed by quantification of the corresponding mRNA.

In some embodiments, the detectable expression of the therapeutic geneproduct in heart tissue occurs at doses, in vector genomes (vg) perkilogram weight of subject (kg), of 3×10¹⁴ vg/kg or less, 2×10¹⁴ vg/kgor less, 1×10¹⁴ vg/kg or less, 9×10¹³ vg/kg or less, 8×10³ vg/kg orless, 7×10¹³ vg/kg or less, 6×10³ vg/kg or less, 5×10¹³ vg/kg or less,4×10¹³ vg/kg or less, 3×10¹³ vg/kg or less, 2×10¹³ vg/kg or less, or1×10¹³ vg/kg or less.

In various embodiments, the compositions described herein contain therAAV virions or vector genomes described herein and one or morepharmaceutically acceptable excipients. Pharmaceutically acceptableexcipients can include vehicles (e.g., carriers, diluents andexcipients) that are pharmaceutically acceptable for a formulationcapable of being injected. These may be in particular isotonic, sterile,saline solutions (monosodium or disodium phosphate, sodium, potassium,calcium or magnesium chloride and the like or mixtures of such salts),or dry, especially freeze-dried compositions which upon addition,depending on the case, of sterilized water or physiological saline,permit the constitution of injectable solutions. Illustrativepharmaceutical forms suitable for injectable use include, e.g., sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions.

In various embodiments, the pharmaceutical compositions of thedisclosure comprise about 1×10⁸ genome copies per milliliter (GC/mL),about 5×10⁸ GC/mL, about 1×10⁹ GC/mL, about 5×10⁹ GC/mL, about 1×10¹⁰GC/mL, about 5×10¹⁰ GC/mL, about 1×10¹¹ GC/mL, about 5×10¹¹ GC/mL, about1×10¹² GC/mL, about 5×10¹² GC/mL, about 5×10¹³ GC/mL, about 1×10¹⁴GC/mL, or about 5×10¹⁴ GC/mL of the viral vector (e.g. rAAV virion).

In various embodiments, the pharmaceutical compositions of thedisclosure comprise about 1×10⁸ viral genomes per milliliter (vg/mL),about 5×10⁸ vg/mL, about 1×10⁹ vg/mL, about 5×10⁹ vg/mL, about 1×10¹⁰vg/mL, about 5×10¹⁰ vg/mL, about 1×10¹¹ vg/mL, about 5×10¹¹ vg/mL, about1×10¹² vg/mL, about 5×10¹² vg/mL, about 5×10¹³ vg/mL, about 1×10¹⁴vg/mL, or about 5×10¹⁴ vg/mL of the viral vector (e.g. rAAV virion).

In some embodiments, the pharmaceutical compositions of the disclosureare administered in a total volume of about 1 mL, 5 mL, 10 mL, about 20mL, about 25 mL, about 30 mL, about 35 mL, about 40 mL, about 45 mL,about 50 mL, about 55 mL, about 60 mL, 65 mL, about 70 mL, about 75 mL,about 80 mL, about 85 mL, about 90 mL, about 95 mL, about 100 mL, about105 mL, about 110 mL, about 115 mL, about 120 mL, about 125 mL, about130 mL, about 135 mL, about 140 mL, about 145 mL, about 150 mL, about155 mL, about 160 mL, about 165 mL, about 170 mL, about 175 mL, about180 mL, about 185 mL, about 190 mL, about 200 mL, about 205 mL, about210 mL, about 215 mL, or about 220 mL.

In some embodiments, the methods of the disclosure compriseadministering an rAAV virion encoding MYBPC3 at a dose of about 1×10⁸genome copies per milliliter (GC/mL), about 5×10⁸ GC/mL, about 1×10⁹GC/mL, about 5×10⁹ GC/mL, about 1·10¹⁰ GC/mL, about 5×10¹⁰ GC/mL, about1×10¹¹ GC/mL, about 5×10¹¹ GC/mL, about 1×10¹² GC/mL, about 5×10¹²GC/mL, about 5×10¹³ GC/mL, about 1×10¹⁴ GC/mL, or about 5×10¹⁴ GC/mL ofthe rAAV virion.

In preferred embodiments, the methods of the disclosure compriseintravenously administering an rAAV virion encoding MYBPC3 at a dose ofabout 3×10¹² GC/mL, about 3×10¹³ GC/mL, about 1×10¹⁴ GC/mL, or about3×10¹⁴ GC/mL of the rAAV virion.

In preferred embodiments, the methods of the disclosure compriseadministering, by localized delivery to the heart, an rAAV virionencoding MYBPC3 at a dose of about 3×10¹¹ GC/mL, about 3×10¹² GC/mL,about 1×10³ GC/mL, or about 3×10¹³ GC/mL of the rAAV virion.

In some embodiments, the methods of the disclosure compriseadministering an rAAV virion encoding MYBPC3 at a dose of about 1×10⁸viral genomes per milliliter (vg/mL), about 5×10⁸ vg/mL, about 1×10⁹vg/mL, about 5×10⁹ vg/mL, about 1×10¹⁰ vg/mL, about 5×10¹⁰ vg/mL, about1×10¹¹ vg/mL, about 5×10¹¹ vg/mL, about 1×10¹² vg/mL, about 5×10¹²vg/mL, about 5×110¹³ vg/mL, about 1×10¹⁴ vg/mL, or about 5×10¹⁴ vg/mL ofthe rAAV virion.

In preferred embodiments, the methods of the disclosure compriseintravenously administering an rAAV virion encoding MYBPC3 at a dose ofabout 3×10¹² vg/mL, about 3×10¹³ vg/mL, about 1×10¹⁴ vg/mL, or about3×10¹⁴ vg/mL of the rAAV virion.

In preferred embodiments, the methods of the disclosure compriseadministering, by localized delivery to the heart, an rAAV virionencoding MYBPC3 at a dose of about 3×10¹¹ vg/mL, about 3×10¹² vg/mL,about 1×10¹³ vg/mL, or about 3×10¹³ vg/mL of the rAAV virion.

Genome copies per milliliter can be determined by quantitativepolymerase change reaction (qPCR) using a standard curve generated witha reference sample having a known concentration of the polynucleotidegenome of the virus. For AAV, the reference sample used is often thetransfer plasmid used in generation of the rAAV virion but otherreference samples may be used.

Alternatively or in addition, the concentration of a viral vector can bedetermined by measuring the titer of the vector on a cell line. Viraltiter is typically expressed as viral particles (vp) per unit volume(e.g., vp/mL). In various embodiments, the pharmaceutical compositionsof the disclosure comprise about 1×10⁸ viral particles per milliliter(vp/mL), about 5×10⁸ vp/mL, about 1×10⁹ vp/mL, about 5×10⁹ vp/mL, about1×10¹⁰ vp/mL, about 5×10¹⁰ vp/mL, about 1×10¹¹ vp/mL, about 5×10¹¹vp/mL, about 1×10¹² vp/mL, about 5×10¹² vp/mL, about 5×10¹³ vp/mL, orabout 1×10¹⁴ vp/mL, or about 5×10¹⁴ of the viral vector (e.g., rAAVvirion).

In one embodiment, the present disclosure provides a kit comprising acontainer housing a pharmaceutical composition as described herein.

The rAAV virions or vector genomes of the present disclosure can beadministered to a subject in need thereof by systemic application, e.g.,by intravenous, intra-arterial or intraperitoneal delivery of a vectorin analogy to what has been shown in animal models (Katz et al., 2012,Gene Ther. 19:659-669. In some embodiments, the rAAV virions or vectorgenomes of the present disclosure treat or prevent hypertrophiccardiomyopathy, wherein the vector is administered systemically.

In some embodiments, the rAAV virions or vector genomes of the presentdisclosure can be delivered by direct administration to the hearttissue, e.g. by intracoronary administration. In some embodiments, thevectors are administered as a single dose by antegrade epicardialcoronary artery infusion over a 10-minute period in a cardiaccatheterization laboratory after angiography (percutaneous intracoronarydelivery without vessel balloon occlusion) with the use of standard 5For 6F guide or diagnostic catheters (Jaski et al., 2009, J Card Fail.15: 171-181).

Subjects who are suitable for treatment using the compositions,compositions and methods of the present disclosure include individuals(e.g., mammalian subjects, such as humans, non-human primates, domesticmammals, experimental non-human mammalian subjects such as mice, rats,etc.) having a cardiac condition.

In some embodiments, the rAAV virions or vector genomes of the presentdisclosure can be used to treat a subject in need thereof. In someembodiments, the viral vector can be administered to the subject in needto treat a cardiovascular disease. In some embodiments, the rAAV virionsor vector genomes are administered to a subject to treat cardiomyopathy.In some embodiments, the viral vector is administered systemically. Inother embodiments, the viral vector is delivered by directadministration to the heart tissue.

rAAV virions or vector genomes can be administered by various routes,including without limitation direct injection into the heart or cardiaccatheterization. In a preferred embodiment, a pharmaceutical compositioncomprising an rAAV virion encoding MYBPC3 is administered byintracardiac catheter delivery via retrograde coronary sinus infusion(RCSI). Alternatively, the viral vector can be administered systemicallysuch as by intravenous infusion. When direct injection is used, it maybe performed either by open-heart surgery or by minimally invasivesurgery. In some cases, the viral vector is delivered to the pericardialspace by injection or infusion.

The viral vector administered to the subject can be traced by a varietyof methods. For example, recombinant viruses labeled with or expressinga marker (such as green fluorescent protein, or beta-galactosidase) canreadily be detected. The recombinant viruses may be engineered to causethe target cell to express a marker protein, such as a surface-expressedprotein or a fluorescent protein. Alternatively, the infection of targetcells with recombinant viruses can be detected by their expression of acell marker that is not expressed by the animal employed for testing(for example, a human-specific antigen when injecting cells into anexperimental animal). The presence and phenotype of the target cells canbe assessed by fluorescence microscopy (e.g., for green fluorescentprotein, or beta-galactosidase), by immunohistochemistry (e.g., using anantibody against a human antigen), by ELISA (using an antibody against ahuman antigen), or by RT-PCR analysis using primers and hybridizationconditions that cause amplification to be specific for RNA indicative ofa cardiac phenotype.

All patents, patent publications, and other publications referenced andidentified in the present specification are individually and expresslyincorporated herein by reference in their entirety for all purposes.

EXAMPLES Example 1: Design of Vector Genome for Large Cargos

The purpose of this study was to evaluate a vector having deletions innon-coding portions of the vector to a parental vector. It demonstratesthat, surprisingly, deletion in non-coding regions increases potency ofthe vector.

With two intact flanking ITR sequences (each ˜130 bp), promoter, intron,WPRE and polyadenylation signal, and standard cis-regulatory sequencesfor optimal transgene expression, typical AAV vector genomes requireabout 1.8-2.0 kbp of non-coding DNA sequence. Trangenes of about 3.0 kbpor greater, like the 3.8 kbp transgene MYBPC3, cause the vector genometo exceed 5.0 kbp. For example, Mearini et al., Nat Commun 5:5515 (2014)reports an AAV vector encoding MYBPC3 having a vector genome size of 5.4kbp. Without ITR sequences, this is about 5.2 kbp.

A reporter system was generated to test whether an AAV vector wouldtolerate shortened non-coding regions. A conventional AAV vector havinga CAG promoter, intron, WPRE, and standard polyA sequence was modifiedto remove the WPRE (589 bp) and to shorten the polyA sequence (removing170 bp) (FIG. 1A). Expression of a GFP reporter cloned into the multiplecloning site (MCS) was maintained but slightly decreased when thesevector elements were deleted or shortened (FIG. 1B). The vector wasfurther truncated by deletion of the intron and a portion of thesequence 3′ to the 5′ ITR (FIG. 1C). The final vector genome was about1.1 kpb (0.8 kpb without ITRs).

Example 2: MYBPC3 Transgene Expression in Induced Cardiomyocytes InVitro

The purpose of this study was to provide an improved tissue-specificpromoter for expression of a therapeutic gene product in inducedcardiomyocytes in vitro using an AAV-based vector system. Human inducedpluripotent stem cells (iPSCs) were differentiated into cardiomyocytesusing mesoderm induction, cardiac specification and metabolic selectionas previously described (Tohyama et al. Cell Stem Cell. 2013;12(1):127-37; Lian et al. Proc Natl Acad Sci USA. 2012;109(27):E1848-57; Burridge P W, Holmstrom A, Wu J C. Curr Protoc HumGenet. 2015; 87:21 3 1-15.) iPSC-CM viral transductions were performedwith AAV6 at the indicated multiplicities of infection.

The gene expression cassettes depicted in FIG. 2A were constructed foran AAV-based vector system for the treatment of cardiomyopathy. The AAVvector, based on the vector depicted in FIG. 1C, comprised severalcis-regulatory elements, including two inverted terminal repeats (ITRs,260 bp each), a polyadenylation signal (A, 49 bp), and a full-length(SEQ ID NO: 1) or modified cardiac troponin T (TNNT2) promoter (SEQ IDNO: 2-4). No WPRE was included, and the polyA signal and the sequence 3′to the 5′ ITR were both shortened. The modified TNNT2 promoterscontained 100-200 bp deletions at the 5′ (upstream) end of the wild-typeTNNT2 promoter. Human myosin binding protein C (MYBPC3, SEQ ID NO: 86)with a polynucleotide sequence length of 3.825 kb was tested as thetherapeutic gene product in iPSC-derived cardiomyocytes.

To determine whether wild-type or modified TNNT2 promoters could inducedetectable expression of the MYBPC3 protein, MYBPC3′ iPSC-derivedcardiomyocytes were transduced with AAV6 particles at 6×10⁴ MOI. Cellswere analyzed for MYBPC3 protein expression by immunofluorescence orWestern blot 5-15 days post-infection.

FIG. 2B shows that the AAV vector comprising the wild-type TNNT2promoter (SEQ ID NO: 1) drives expression of MYBPC3 protein in thesarcomeres of MYBPC3′ iPSC-derived cardiomyocytes.

FIG. 3A-FIG. 3C show that the AAV vector comprising the 400 bp modifiedTNNT2 promoter (SEQ ID NO: 3) drives higher MYBPC3 protein expressionthan either the 600 bp wild-type TNNT2 (SEQ ID NO: 1) or 500 bp modifiedTNNT2 (SEQ ID NO: 2) promoters in transduced MYBPC3^(−/−) iPSC-derivedcardiomyocytes. In contrast, MYBPC3^(−/−) iPSC-derived cardiomyocytestransfected with a plasmid (rather than transduced with virus) encodingMYBPC3 under the control of either the 600 bp wild-type TNNT2 (SEQ IDNO:1) or 400 bp modified TNNT2 promoter (SEQ ID NO: 3) showed similarMYBPC3 protein expression.

Example 3: Mybpc^(−/−) Mice Model Hypertrophic Severe Cardiomyopathy

Homozygous Mybpc3 knockout mice (KO) were generated on a C57Bl/6background by a CRISPR-Cas9 paired gRNA deletion of exons one and two(FIG. 4A). KO mice exhibited severe deficits in cardiac function (FIG.4C and FIG. 4D) and pronounced cardiac hypertrophy (FIGS. 4E-4G) at twoweeks of age, despite normal Mendelian ratios and comparable body weightto wild-type littermates (FIG. 4B). This model has more severe cardiachypertrophy than other models (Schlossarek et al. Basic Res. Cardiol.107:1-13 (2012)). Our KO mice exhibit severe, early-onset HCM injuveniles (two-week-old mice) that models pediatric onset of HCM inhumans, as well as late-stage HCM in adults. See Lekanne Deprez et al.,J Med Genet 43:829-832 (2006); Xin et al., Am J Med Genet Part A143A:2662-2667 (2007); Zahka et al., Heart 94:1326-1330 (2008);Marziliano et al., Neonatology 102:254-258 (2012); Wessels et al., Eur JHum Genet 23:922-928 (2015).

Example 4: MYBPC3 Transgene Expression in Heart Tissue In Vivo

The purpose of this study was to examine therapeutic protein expressionin vivo using an AAV-based vector system comprising a modifiedcardiac-specific promoter.

Adult mice were retro-orbitally injected with AAV9 recombinant viruscomprising either the 600 bp wild-type TNNT2 (SEQ ID NO: 1) or 400 bpmodified TNNT2 (SEQ ID NO: 3) promoter operatively linked to apolynucleotide that encodes MYBPC3. Tissue samples from heart, skeletalmuscle (tibialis anterior), liver and whole brain were harvested 2 weekspost-infection. RNA was extracted from all tissues, synthesized to cDNAand analyzed by qRT-PCR using primers specific to human MYBPC3.

As shown in FIG. 5, mice injected with the AAV9-based vector comprisingeither the wild-type or modified TNNT2 promoter showed high levels ofMYBPC3 mRNA in heart tissue compared to skeletal, brain or liver tissue.The 400 bp modified TNNT2 promoter showed increased expression of MYBPC3mRNA in heart tissue compared to the 600 bp wild-type TNNT2 promoter.

As shown in FIGS. 6A-6B, adult mice were intravenously dosed via tailvein injection with an AAV9 vector with the 400 bp modified TNNT2promoter cassette. Tissue samples from heart and liver were harvested 4weeks post-injection. Absolute quantification of viral genomes permicrogram of genomic DNA was assessed by qPCR using linearizedstandards. RNA was extracted from all tissues, synthesized to cDNA andanalyzed by qRT-PCR using primers specific to human MYBPC3.Surprisingly, the 400 bp TNNT2 promoter retains high selectivity for theheart: despite the 100-fold greater vector genomes detected in the liverthan the heart 4 weeks post-injection in the adult-dosed animals (FIG.6A, logarithmic scale), liver expression of the transgene was less than1/10,000th of cardiac expression (FIG. 6B, logarithmic scale).

Collectively, these results indicate that a 200 bp deletion from thewild-type TNNT2 promoter, i.e., the 400 bp modified TNNT2 promoter (SEQID NO: 3), effectively drives expression of MYBPC3 protein incardiomyocytes with high selectivity despite deletion of a substantialportion of the promoter sequence.

Example 5: Rescue of Cardiac Function in Mybpc3 Null Mice

This example demonstrates functional rescue of loss of function in theMybpc3 in mice, using the vector designed for large cargoes described inExample 1 and the 400 bp modified hTNNT2 promoter described in Examples2 and 3.

The 400 bp hTNNT2 promoter and murine Mybpc3 gene were cloned into thevector shown in FIG. 1C. This vector was packaged using an AAV9 capsidto generate the test vector.

In a first experiment, homozygous Mbpc3^(−/−) mice were injectedretro-orbitally at two weeks of age with 1E14 vg·kg⁻¹ test vectorencoding Mybpc3 or vehicle, HBSS. Cardiac tissue was harvested two weekslater (at four weeks), along with that of wild-type littermates. Theexperimental vector achieved wild-type levels of MYBPC3 proteinexpression in Mybpc3^(−/−) mice at two weeks post-injection (FIG. 7). Weconclude the test vector was capable of expressing MYBPC3 atphysiological levels in juvenile animals at a dose as low as 1E14vg·kg⁻¹

In a second experiment, the first experiment was repeated at a lowerdose, 3E13 vg·kg⁻¹. Homozygous Mybpc3^(−/−) mice were injectedretro-orbitally at two weeks of age with 3E13 vg·kg⁻¹ and 1E14 vg·kg⁻¹test vector encoding Mybpc3 or vehicle, HBSS. Wild-type levels ofcardiac MYBPC3 protein expression were detected by ELISA in Mybpc3^(−/−)mice at two and six weeks post-injection (FIG. 8) We conclude the testvector was capable of expressing MYBPC3 at physiological levels injuvenile animals at a dose as low as 3E13 vg·kg⁻¹

In a third experiment, cardiac function was assessed using assaysrelevant to hypertrophic cardiomyopathy. Hypertrophic cardiomyopathypresents physiologically as (1) an increase in heart size, measured asreported as the ratio of left ventricular mass/total body mass (LVM/BW)in mg·g⁻¹; and (2) as fractional shortening (FAS), measured byechocardiography.

Homozygous Mybpc3^(−/−) mice were injected retro-orbitally at two weeksof age with 1E13 vg·kg⁻¹, 3E13 vg·kg⁻¹, and 1E14 vg·kg⁻¹ of test vectorencoding Mybpc3 or vehicle, HBSS. Dose-dependent rescue of cardiacfunction was observed at all tested doses (1E13 vg·kg⁻¹, 3E13 vg·kg⁻¹,and 1E14 vg·kg⁻¹). LVM/BM was decreased from vehicle control at alltested doses six weeks post-injection (FIG. 9A). FAS (expressed as %percentage change in LV internal dimensions between systole anddiastole, FIG. 9B) and ejection fraction (FIG. 9C) were increased fromvehicle control at all tested doses six weeks post-injection. Evengreater improvements in LVM/BM (FIG. 9D), FAS (FIG. 9E), and EF (FIG.9F) were observed 31 weeks following injection. Consistent with equallevels of MYBPC3 protein expression observed at 3E13 vg·kg⁻¹ and 1E14vg·kg⁻¹ doses (see FIG. 8), animals treated with 3E13 vg·kg⁻¹ or 1E14vg·kg⁻¹ exhibit similar improvements in hypertrophy, FAS or EFimprovements. Even at only 1E13 vg·kg⁻¹ dose, hypertrophy, FAS and EFare all improved compared to vehicle control.

We conclude the test vector was capable of rescue of cardiac function injuvenile animals at a dose as low as 1E13 vg·kg⁻¹1.

Rescue of function in symptomatic juvenile mice is, in the case ofhypertrophic cardiomyopathy, more challenging than prevention of declinein function in infants, because hypertrophic cardiomyopathy is aprogressive disorder. Older animals exhibit more severe disease thanyounger animals. To our knowledge, rescue of MYBPC3 loss of function insymptomatic juvenile animals has never been demonstrated before withAAV. Our model is also a complete loss of function caused by deletion ofthe Mybpc3 gene, not a partial loss of function due to mutation.

We compared our results to those reported in Mearini et al., Nat.Commun. 5:5515 (2014), which used a 5.4 kb expression cassette encodingMybpc3 in mice having a single nucleotide polymorphism in the endogenousMybpc3 gene. Mearni et al. report prevention of high LVM/BW at two-weeksof age in mice injected as neonates (not symptomatic juveniles) withvery high doses (1E12 vg and 3E12 vg, which corresponds to 7E14 vg·kg⁻¹and 2E15 vg·kg⁻¹, based on an average neonate mass of 1.5 g) of an AAV9vector encoding the same Mybpc3 gene. Mearni et al. used a 550 bp hTNNT2promoter, rather than the present 400 bp modified hTNNT2 promoter. Thevector Mearini et al. does not significantly prevent FAS decline, evenat 2E15 vg·kg⁻¹. By contrast, the present vector demonstratesimprovement in physiological parameters in juveniles animals (not onlyneonates) at doses at least as low as 1E13 vg·kg⁻¹ to 1E14 vg·kg⁻¹.

The vector and promoter modifications dramatically and surprisinglyincrease potency of the vector.

Example 6: Direct Comparison of 5.4 Kbp Cassette to 4.7 Kbp Cassette

This example directly compares a 5.4 kbp cassette encoding the Mybpc3gene to a 4.7 kbp cassette encoding the Mybpc3 gene in mature (2.5months of age) homozygous mice with advanced disease.

Homozygous Mybpc^(−/−) mice were injected retro-orbitally with 3E13vg·kg⁻¹ or 1E14 vg·kg⁻¹ of AAV9 vector encoding Mybpc3 in the context ofthe 5.4 kbp or 4.7 kbp cassettes (FIG. 10A), or injected with vehiclecontrol, HBSS. Even when dosed at this advanced stage of cardiacdecline, the 4.7 kbp cassette significantly improved cardiac functionbased on ejection fraction (EF) (FIG. 10B), with clear restoration offunction above pre-dose baseline (FIG. 10C), unlike animals treated withvehicle (Veh) or the 5.4 kbp cassette. Further, compared to the 5.4 kbpcassette, the 4.7 kbp cassette was also able to significantly decreasehypertrophy, as indicated by LVM/BM, eighteen weeks post-injection (FIG.10D).

This example demonstrates functional rescue of loss of function in theMybpc3 in mice, using the vector backbone and promoter modificationsdescribed in Examples 1-3.

This example also demonstrates that, in a challenging model ofdisease—adult, homozygous Mybpc3^(−/−) mice—a 5.4 kbp vector (SEQ ID NO:201) at low dose fails to generate any physiological improvement;whereas a 4.7 kbp vector according to the present disclosure causesstatistically significant improvement in physiological parametersrelated to cardiomyopathy at doses as low as 3E13 vg·kg⁻¹.

Example 7: Greater Efficacy with an Improved AAV Capsid Encoding MYBPC3

This example demonstrates how the improved potency of the large cargovector (Example 1) and modified promoter (Examples 2 and 3) based onrescue of Mybpc3^(−/−) mice (Example 5) can be further improved by useof an engineered AAV capsid.

An AAV9 capsid variant, CR9-10 exhibited significantly higher cardiactransduction upon systemic delivery in adult mice than AAV9 with aGFP-encoding cassette as determined by ELISA (p<0.05, One-way ANOVA;Dunnett's multiple comparison test) (FIG. 11A).

In a second experiment, the expression cassette encoding the murineMybpc3 gene was packaged into either AAV9 or CR9-10 and the potency ofcardiac rescue in Mybpc^(−/−) mice compared. Homozygous Mybpc^(−/−) micewere injected retro-orbitally at two weeks of age with 1E13 vg·kg⁻¹ and3E13 vg·kg⁻¹ of AAV9 vector, CR9-10 vector, or vehicle control HBSS. Alltest articles significantly improved cardiac function based on ejectionfraction (EF) (FIG. 11B), with clear restoration of function abovepre-dose baseline (AEF)(FIG. 11C). Consistent with improved cardiactransduction, CR9-10 resulted in greater EF improvement than AAV9.

Example 8: Non-Human Primate Study of Engineered AAV Capsid Variants

Biodistribution of AAV vectors having engineered capsids (described inU.S. Provisional Patent Appl. No. 63/012,703, which is incorporatedherein in its entirety), were assessed in male cynomolgus macaques(Macaca fascicularis) following intravenous delivery.

AAV vector generated with fourteen different capsids, including AAV9,were pooled and injected into NHPs at a 1E13 vg·kg-1 dose (n=3). ViralDNA was extracted from left ventricle and liver one month after systemicdelivery. Consistent with the murine results, CR9-10 exhibited increasedcardiac transduction compared to AAV9 (FIG. 12A). Additionally, manyvariants decreased liver viral burden relative to AAV9 (FIG. 12B),improving the ratio of left ventricle transduction to liver infection(FIG. 12C).

Example 10: Rescue of Cardiac Function with the Human MYBPC3 Gene inMybpc3 Null Mice

This example demonstrates the ability of human MYBPC3 gene to rescueMybpc3^(−/−) mice.

Homozygous Mybpc3^(−/−) mice were injected retro-orbitally at two weeksof age with AAV9 encoding the mouse Mybpc3 gene (mMybpc3) (at 1E14vg·kg-1), AAV9 encoding the human MYBPC3 gene (hMYBPC3) (at 1E14vg·kg-1), or vehicle, HBSS. Cardiac size and function were monitored byechocardiography up to eight months post-injection. The results indicatethat AAV9-mediated cardiac MYBPC3 replacement of either mMybpc3 andhMYBPC3 in Mybpc^(−/−) mice resulted in recovery of cardiac size andfunction. Ejection fraction (EF) was significantly improved by both thehuman and mouse orthologs of MYBPC3, with mMybpc3 yielding greaterimprovement in EF compared to hMYBPC3 (FIG. 13A). Importantly, hMYBPC3was just as potent as mMybpc3 at reducing cardiac hypertrophy over time,as evidenced by left ventricular mass normalized to body weight (LVM/BW)(FIG. 13B). This was further validated by comparable decreases in leftventricular posterior wall thickness during diastole (LVPW;d) (FIG.13C). Critically, all improvements exhibited robust stability out to 8months post-injection.

In a second study, the efficacy of hMYBPC3 was assessed over a range ofviral doses. Homozygous Mybpc3^(−/−) mice were injected retro-orbitallyat two weeks of age with 1E13 vg·kg⁻¹, 1E14 vg·kg-1, and 3E14 vg·kg-1 oftest vector encoding the human MYBPC3 gene or vehicle, HBSS.Dose-dependent improvement of cardiac function was observed for alltested doses (1E13 vg·kg-1, 1E14 vg·kg-1, and 3E14 vg·kg-1) fourteenweeks post-injection, as indicated by EF (FIG. 14A), with significantimprovement above pre-dose baseline for 1E14 vg·kg-1 and 3E14 vg·kg-1treatments (FIG. 14B). Significant reduction in cardiac hypertrophy wasalso observed for 1E14 vg·kg-1 and 3E14 vg·kg-1 treatments, based onLVM/BW (FIG. 14C). Thus, we conclude the hMYBPC3 test vector was capableof preserving cardiac function in adult animals at a dose as low as 1E13vg·kg-1.

Example 11: Treatment of Hypertrophic Cardiomyopathy (HCM)

Cardiomyopathy is the number-one cause of sudden cardiac arrest inchildren under 18. Hypertrophic cardiomyopathy (HCM) affects 0.5 millionAmericans, potentially resulting in heart failure or sudden death.Loss-of-function mutations in Myosin Binding Protein C3, MYBPC3, are themost common genetic cause of HCM. The majority of MYBPC3 mutationscausative for HCM result in truncations, via nonsense, frameshift orsplice-site mutations. The sarcomeric pathophysiology of the majority ofHCM patients with MYBPC3 mutations appears to be due tohaploinsufficiency, as the total amount of MYBPC3 protein incorporatedinto sarcomeres falls significantly below normal. Decreased sarcomericlevels of MYBPC3 result in decreased myosin inhibition with more myosinheads engaged on the actin filament, resulting in hypercontractility.

The clearest path to the treatment of haploinsufficiency is therestoration of the insufficient gene product; in this case wild-typeMYBPC3. Thus, we have successfully engineered an AAV vector (TN-201)with superior properties for selective restoration of MYBPC3 tocardiomyocytes upon systemic delivery. Critically, we have demonstratedfor the first time with AAV the ability of both a mouse surrogate andTN-201 to reverse cardiac dysfunction and hypertrophy in a symptomaticmurine model of disease.

Dose-ranging efficacy studies exhibited restoration of wild-type MYBPC3protein levels and saturation of cardiac improvement at the clinicallyrelevant dose of 3E13 vg/kg. Further, pilot safety studies in adult andinfant mice injected with >10× an efficacious dose exhibited no clinicalobservations, no alterations in cardiac function, and nohistopathological findings. Importantly, we have determined that TN-201produced utilizing the highly scalable Sf9 platform results in similarlypotent efficacy in a Mybpc3 model of disease. Finally, we haveestablished that our observed efficacy is sufficiently meaningful forstable benefit up to 8 months post-injection, as well as reversal ofcardiac dysfunction even in late-stage homozygote disease.

Example 12: Clinical Studies

A pharmaceutical composition comprising rAAV virions encoding MYBPC3, asdescribed herein, is administered by intravenously or by retrogradecoronary sinus (RCSI). Functional efficacy is determined by cardiacfunctional status assessments (e.g., New York Heart AssociationFunctional Classification, NYHA; Cardiopulmonary exercise test, CPET),quality of life questionnaires (e.g., Kansas City CardiomyopathyQuestionnaire Clinical Quality Score, KCCQ-CSS), cardiac imaging (e.g.,echocardiography), cardiac biomarkers (e.g. troponin and NT-proBNP),cardiac rhythm and immunologic assessments, cardiac functional statusassessments (e.g., Pediatric Interagency Registry for MechanicallyAssisted Circulatory Support, PEDIMACS; Ross classifications), and/orMajor Adverse Cardiac Events (MACE) (total death, cardiactransplantation, initiation of inotropes, initiation of ventilatory, ormechanical circulatory support). Clinical studies may include monitoringsafety and continued efficacy (e.g., adverse events, severe adverseevents, electrocardiogram, cardiac enzymes, biomarkers, functionalstatus, left ventricular (LV) function/mass, quality of life, serumchemistries, liver function tests) on an annual basis for up to 10years.

1. A method of treating a cardiomyopathy caused by a MYBPC3 mutation ina subject in need thereof, comprising administering an effective amountof a recombinant adeno-associated virus (rAAV) virion, comprising avector genome comprising an expression cassette comprising apolynucleotide encoding MYBPC3 operatively linked to a promoter, theexpression cassette flanked by a 5′ inverted terminal repeat (ITR) and a3′ ITR; wherein the vector genome comprises at most 4.8 kbp.
 2. Themethod of claim 1, wherein the promoter is a cardiac troponin Tpromoter.
 3. The method of claim 2, wherein the cardiac troponin Tpromoter comprises about 400 bp.
 4. The method of claim 2, wherein thecardiac troponin T promoter comprises about 400 bp and wherein thecardiac troponin T promoter shares at least 90% identity to thepolynucleotide sequence of SEQ ID NO:
 3. 5. The method of claim 1,wherein MYBPC3 shares at least 90% identity to the amino acid sequenceof SEQ ID NO:
 103. 6. The method of claim 1, wherein the polynucleotideencoding MYBPC3 shares at least 80% identity to the polynucleotidesequence of SEQ ID NO:
 86. 7. The method of claim 1, wherein the vectorgenome comprises at most 4.7 kbp.
 8. The method of claim 1, wherein thevector genome comprises 4.7 kbp.
 9. The method of claim 1, wherein the5′ ITR comprises a sequence that shares 95% identity to thepolynucleotide sequence of SEQ ID NO: 96; and the 3′ ITR comprises asequence that shares at least 95% identity to the polynucleotidesequence of SEQ ID NO:
 97. 10. The method of claim 2, wherein thecardiac troponin T promoter shares at least 80% identity to thepolynucleotide sequence of SEQ ID NO:
 3. 11. The method of claim 2,wherein the cardiac troponin T promoter shares 95% identity to thepolynucleotide sequence of SEQ ID NO:
 3. 12. The method of claim 1,wherein the expression cassette shares at least 80% identity to thepolynucleotide sequence of SEQ ID NO:
 95. 13. The method of claim 1,wherein the expression cassette shares 90% identity to thepolynucleotide sequence of SEQ ID NO:
 95. 14. The method of claim 1,wherein the vector genome shares at least 80% identity to thepolynucleotide sequence of SEQ ID NO:
 102. 15. The method of claim 1,wherein the vector genome shares 90% identity to the polynucleotidesequence of SEQ ID NO:
 102. 16. The method of claim 1, wherein the rAAVvirion is a serotype AAV9 virion.
 17. The method of claim 1, wherein thecardiomyopathy is hypertrophic cardiomyopathy.
 18. The method of claim17, wherein the method increases ejection fraction in the subject. 19.The method of claim 1, wherein the MYBPC3 mutation is a homozygousMYBPC3^(−/−) mutation.
 20. A method of expressing MYBPC3 protein in acell, comprising transducing the cell with a recombinantadeno-associated virus (rAAV) virion, comprising a vector genomecomprising an expression cassette comprising a polynucleotide encodingMYBPC3 operatively linked to a promoter, the expression cassette flankedby a 5′ inverted terminal repeat (ITR) and a 3′ ITR; wherein the vectorgenome comprises at most 4.8 kbp.
 21. The method of claim 20, whereinthe cell is a cardiac cell.
 22. The method of claim 20, wherein thevector genome comprises 4.7 kbp.
 23. The method of claim 20, wherein thepromoter is a cardiac troponin T promoter sharing at least 95% identityto the polynucleotide sequence of SEQ ID NO.
 3. 24. The method of claim20, wherein the vector genome shares at least 95% identity to thepolynucleotide sequence of SEQ ID NO:
 102. 25. The method of claim 20,wherein the rAAV virion is a serotype AAV9 virion.